TY - JOUR
AU - Huh, Nam-ho
AB - Abstract Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, ∼24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1α, and HNF4α. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases. Disclosure of potential conflicts of interest is found at the end of this article. Rat bone marrow, Clonal stem cell lines, Hepatocyte-like cells, Mass production, Intrasplenic transplantation Introduction The most effective therapy for acute or chronic hepatic failure is orthotopic liver transplantation. However, serious shortage of donor organs limits applicability of this therapy. Although hepatocyte transplantation could be an alternative therapy, the donor shortage limits this approach as well unless a method for vast propagation of normal human hepatocytes in a safe and reliable manner will be developed. Tissue stem cells are another potential source of functional hepatocytes because of their multipotency, essentially unlimited proliferation capacity, and relative easiness for preparation. Among tissues and organs from which stem cells have been isolated [1–6], bone marrow is the most fertile [7, 8]. Hematopoietic stem cells (HSCs) [9], mesenchymal stem cells (MSCs) [10], and multipotent adult progenitor cells (MAPCs) [8, 11, 12] have been isolated from the bone marrow. In addition, a variety of cells have also been isolated from the adherent stromal layer of the bone marrow with putative functional mesenchymal stem cell attributes [13, 14]. At present, it is unclear whether they may be different or represent subsets of the same mesenchymal stem cell. Most of these stem cells or stem-like cells have been demonstrated to differentiate into hepatocytes in vivo or in culture [8–14]. We previously have shown that rat bone marrow-derived cells with HSC markers could be induced to show hepatocyte phenotypes in culture by treatment with hepatocyte growth factor (HGF) [15–17]. These observations implied possible therapeutic application of such cells against liver failure. However, the limited growth capacity and inefficiency in differentiation of the stem cells have hampered the preparation of highly functional hepatocyte-like cells in a large quantity, this being a prerequisite for practical application. In the present study, therefore, we aimed at isolating stem cells from bone marrow of adult rats that can overcome these problems. Materials and Methods Cytokines, Matrix Proteins, and Enzymes Epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-BB (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and fibroblast growth factor (FGF)-4 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) were commercially obtained. HGF (Δ5 variant, a mature two-chain form) was kindly provided by Daiichi-Sankyo (Tokyo, http://www.daiichisankyo.co.jp) [18]. Fibronectin and Matrigel were purchased from Sigma-Aldrich and BD Biosciences (San Diego, http://www.bdbiosciences.com), respectively. Type I collagen was prepared from rat-tail tendon as described previously [16]. Trypsin (1:250) and type I collagenase were purchased from Sigma-Aldrich, respectively. Medium Composition MAPC medium consisted of the following: 60% Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical, Tokyo, http://www.nissui-pharm.co.jp/index_e.html), 40% MCDB-201 (Sigma-Aldrich), 1× insulin-transferrin-selenium-A containing sodium pyruvate (Gibco-BRL, Gaithersburg, MD, http://www.invitrogen.com), 1× linoleic acid bovine serum albumin (Sigma-Aldrich), 10−9 M dexamethasone (Banyu Pharmaceutical, Tokyo, http://www.banyu.co.jp), 10−4 M ascorbic acid 2-phosphate (Sigma-Aldrich), penicillin (100 units/ml), streptomycin (100 μg/ml), and 2% fetal bovine serum (FBS) [8]. Cultivation of Bone Marrow Cells, Single Cell Cloning, and Transfection of Green Fluorescent Protein Gene Whole bone marrow cells were isolated from the femora of 8-week-old Sprague-Dawley female rats and green fluorescent protein (GFP)-transgenic Wistar male rats and seeded at 8 × 105 cells per 0.2 ml/cm2 into collagen-coated dishes containing HGM medium [19] supplemented with HGF (1 μg/ml) and EGF (20 ng/ml) as described previously [16]. For subculture, cells were detached by treatment with 0.2% trypsin and 0.02% EDTA and transferred at 2.5 × 103 cells per 0.2 ml/cm2 into fibronectin (1 μg/cm2)-coated dishes containing MAPC medium supplemented with EGF (10 ng/ml) and PDGF-BB (10 ng/ml) [8]. Thus, cells were continuously subcultured at an interval of 3 or 4 days. Single cell cloning was performed by the limiting dilution method, and several clonal cell lines were established. Some cell lines were transfected with the plasmid pEGFP-N2 (BD Biosciences) with TransIT-LT1 reagent (Mirus, Madison, WI, http://www.mirusbio.com). After G418 treatment, GFP-expressing cells were isolated by fluorescence-activated cell sorting (FACS). Chromosome Analysis Chromosome preparations with G-banding were made under conventional conditions, and karyotypes were determined according to the rat chromosome idiogram [20]. FACS Analysis Cells were stained with the following antibodies: fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against rat CD29, CD44, CD49b (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), and CD90 (eBioscience Inc., San Diego, http://www.ebioscience.com) or phycoerythrin (PE)-conjugated monoclonal antibody against rat CD45 (eBioscience). Cells were also stained with FITC- or PE-labeled isotype-matched immunoglobulins and used as negative controls. The stained cells were analyzed using a FACSCalibur cytometer and CellQuest software (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Induction of Differentiation into Hepatocytes Cells were inoculated at 2.5 × 103 cells per cm2 into 1% Matrigel-coated dishes containing MAPC medium supplemented with HGF (20 ng/ml) and FGF-4 (10 ng/ml) and cultured for 7 days [11]. Induction of Osteogenic Differentiation Cells were inoculated at 2.5 × 103 cells per cm2 on collagen substratum in DMEM supplemented with 10% FBS, 0.1 μM dexamethasone, 50 μM ascorbic acid 2-phosphate, and 10 mM β-glycerophosphate and cultured for 3 weeks [21]. Cell Transplantation Cells were suspended in phosphate-buffered saline at 7 × 107 cells per 0.7 milliliter and transplanted into the spleen of syngeneic rats (8 weeks old). No immunosuppressive reagent was given after the cell transplantation (CTx). 90% Hepatectomy Ninety percent hepatectomy was performed by removing the median, left lateral, and right upper and lower lobes, leaving only the caudate lobe, as previously described [22]. All of the experiments were performed in conformity to the institutional code for animal experiments. Measurement of Blood Ammonia Level Blood samples were collected from living rats or rats within 1 hour after death. Blood ammonia levels were determined using Fuji DRI-CHEM 100N according to the manufacturer's protocol (FUJIFILM, Tokyo, http://www.fujifilm.com). Immunostaining of Cells and Tissues Cultured cells or frozen tissue sections (6 μm in thickness) were fixed with 4% paraformaldehyde and treated with a primary antibody, that is, rabbit anti-human albumin (Dako, Glostrup, Denmark, http://www.dako.com), goat anti-human hepatocyte nuclear factor (HNF)1α (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), rabbit anti-human HNF4α (Santa Cruz), or mouse anti-GFP antibody (Clontech, Palo Alto, CA, http://www.clontech.com). Some cells were fixed with methanol and treated with anti-human vimentin (Dako) or anti-human fibronectin (Chemicon, Temecula, CA, http://www.chemicon.com). For isotype controls, cells and tissues were treated with rabbit, goat, or mouse IgG instead of respective specific antibodies. These samples were further treated with a second antibody, Alexa Fluor 594- and 488- (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), FITC-, or tetramethylrhodamine isothiocyanate-conjugated antibody (Sigma-Aldrich) and mounted with Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Oil Red O Staining Frozen liver sections (6 μm in thickness) were fixed with 4% paraformaldehyde, stained with oil red O, and counterstained with hematoxylin. Isolation of Liver Cells Liver cells were isolated from rats by two-step collagenase liver perfusion [23] and used for genomic polymerase chain reaction (PCR) and Western blot analysis of GFP gene and its protein. Reverse Transcription-PCR and Northern Blot Analysis Total RNA was isolated by the guanidine-thiocyanate-phenol method, and 1 μg of RNA was used for cDNA synthesis. The resulting products were amplified under the following conditions: initial incubation at 94°C for 4 minutes followed by 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds and then a final step at 72°C for 5 minutes. The primers and expected lengths of products were as follows: Albumin (503 base pairs [bp]): forward, 5′-gacaacattcctgccgatct-3′; reverse, 5′-agcacacacagacggttcag-3′ Glucose 6-phosphatase (G6Pase; 550 bp): forward, 5′-tctaccttgcggctcacttt-3′; reverse, 5′-ttgtgtgtctgtcccaggag-3′ Triptophan-2,3-dioxygenase (TO; 498 bp): forward, 5′-gagcaggagcagacgctatt-3′; reverse, 5′-caccttgtacctgtcgctca-3′; nested (389 bp): forward, 5′-aacgcacacctggcttagag-3′; reverse, 5′-cttgctgcctagcatcctgt-3′ Tyrosine aminotransferase (TAT; 492 bp): forward, 5′-gtccatcggctacctatcca-3′; reverse, 5′-caggacaggatgggaacatt-3′; nested (457 bp): forward, 5′-gggaggaggtcgcttcttac-3′; reverse, 5′-ggaacattggtgctgaggtt-3′ Cytochrome P450 (CYP1A1; 419 bp): forward, 5′-aagtgcagatgcggtcttct-3′; reverse, 5′-cacctccgtgccagtatttt-3′ CYP1A2 (438 bp): forward, 5′-ggggaagaacccacacctat-3′; reverse, 5′-atggctccgatgacattagc-3′ Tumor necrosis factor-α (423 bp): forward, 5′-ccgatttgccatttcatacc-3′; reverse, 5′-cgtgtgtttctgagcatcgt-3′ HGF (487 bp): forward, 5′-tcctgtgccaaaacaaaaca-3′; reverse, 5′-ggtgctgactgcatttctca-3′ Transforming growth factor-α (TGF-α; 253 bp): forward, 5′-cagcatgtgtctgccactct-3′; reverse, 5′-caggcagtccttcctttcag-3′ c-Met (728 bp): forward, 5′-cagtgatgatctcaatgggcaat-3′; reverse, 5′-aatgccctcttcctatgacttc-3′ EGF receptor (EGFR; 376 bp): forward, 5′-gagctcgtggaacctctcac-3′; reverse, 5′-gggagccaatgttgtcctta-3′ Stromal cell-derived factor-1 (SDF-1; 270 bp): forward, 5′-atggacgccaaggtcgtcgc-3′; reverse, 5′-ttacttgtttaaggctttgt-3′ Chemokine (C-X-C motif) receptor 4 (CXCR4; 525 bp): forward, 5′-gcaatgggttggtaatcctg-3′; reverse, 5′-tggagtgtgacagcttggag-3′ Alkaline phosphatase (823 bp): forward, 5′-acctcatcagcatttggaagagct-3′; reverse, 5′-gaacagggtgcgtagggggaacag-3′ Osteonectin (874 bp): forward, 5′-gtgccgagagttcccagcatcatg-3′; reverse, 5′-attcctccagggcaatgtacttgt-3′ Osteopontin (814 bp): forward, 5′-cttgcctcctgtctcccggtgaaa-3′; reverse, 5′-tgtgaaactcgtggctctgatgtt-3′ Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; 440 bp): forward, 5′-atgggaagctggtcatcaac-3′; reverse, 5′-ggatgcagggatgatgttct-3′. GAPDH was used as an internal control. The amplified products were subjected to electrophoresis in 1% agarose gels and stained with ethidium bromide. Northern blot analysis was carried out essentially as previously reported [24]. The PCR product of GAPDH described above was used as a probe. Other probes were also prepared by reverse transcription (RT)-PCR under the same conditions. The used primers and expected lengths of products were as follows: Peroxisome proliferator activated receptor gamma (PPARγ; 505 bp): forward, 5′-gaccactcccattcctttga-3′; reverse, 5′-ccaacagcttctccttctcg-3′ Fatty acid synthase (FAS; 556 bp): forward, 5′-cctgagggccctaccttaac-3′; reverse, 5′-ggctctggtggcttctagtg-3′ Lipoprotein lipase (LPL; 526 bp): forward, 5′-aacattggagaagccattcg-3′; reverse, 5′-ctgaccagcggaagtaggag-3′ Adipocyte lipid-binding protein (ALBP; 445 bp): forward, 5′-tggaaactcgtctccagtga-3′; reverse, 5′-aaaccaccaaatcccatcaa-3′ Adipsin (480 bp): forward, 5′-atggatggagtgaccaagga-3′; reverse, 5′-agatccccacgtaaccacag-3′ Resistin (407 bp): forward, 5′-tgtcactgtgtcccatggat-3′; reverse, 5′-tccagaccctcatctcgttt-3′. The signal intensities of Northern blots were quantified using ImageQuant version 3.3 (Molecular Dynamics, Sunnyvale, CA, http://www.mdyn.com). Genomic PCR Genomic DNA was extracted using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, http://mbbnet.umn.edu/company_folder/gsi.html) according to the manufacturer's protocol. Conditions of PCR and primers are as follows: GFP (302 bp): forward, 5′-cacaagctggagtacaactac-3′; reverse, 5′-tacttgtacagctcgtccatg-3′ β-Actin (380 bp): forward, 5′-ttcaacaccccagccatgtac-3′; reverse, 5′-tcattgccgatagtgatgacc-3′. The amplification was carried out under the following conditions: initial incubation at 94°C for 3 minutes followed by 30 (for β-actin) or 40 cycles (for GFP) at 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 20 seconds and then a final step at 72°C for 7 minutes. For other details, see RT-PCR. Western Blot Analysis Western blot analysis for albumin, HNF1α, HNF4α, or GFP was performed as previously described [25] using the same antibodies utilized for immunostaining and horseradish peroxidase-conjugated anti-rabbit, goat, or mouse IgG antibody (MBL International Corp., Woburn, MA, http://www.mblintl.com). Results Isolation and Characterization of Clonal Stem Cell Lines from the Bone Marrow of Adult Rats Whole bone marrow cells from adult rats were inoculated on collagen substratum in HGM medium supplemented with 10% FBS, HGF (1 μg/ml), and EGF (20 ng/ml) as previously described [16]. Four to six weeks later, flat spindle-shaped cells appeared and grew gradually, and many of those cells accumulated lipid droplets in the cytoplasm (Fig. 1A-1). On culture day 66, the cells were transferred to fibronectin-coated dishes containing MAPC medium supplemented with EGF (10 ng/ml) and PDGF-BB (10 ng/ml) [8]. The cells were subcultured at first once a week and afterward once in 3∼4 days. Approximately 6 weeks after the first subcultivation, small spindle/asteroid cells became gradually predominant. At day 192 (117 population doubling levels [PDLs]), the cells were cloned by a limiting dilution method, and one of the typical clones (rBM25/S3) was further characterized. Figure 1. Open in new tabDownload slide Establishment of a clonal cell line (rBM25/S3) from the bone marrow of an adult female rat. Isolation process of rBM25/S3 cells. Primary culture at day 66 (just before the first subcultivation) showing cells with lipid droplets in the cytoplasm (A-1); small asteroid rBM25/S3 cells established by a limiting dilution method at day 192 (PDL 117) (A-2). Bars indicate 50 μm (A). rBM25/S3 cells rapidly grew with a doubling time of 24 hours for at least 300 PDLs over a period of 300 days on fibronectin-coated dishes in the presence of EGF (10 ng/ml) and PDGF-BB (10 ng/ml). Arrow indicates the timing of the single cell cloning (B). Indispensability of EGF and PDGF-BB for the growth of the cells (C). Abbreviations: EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PDL, population doubling level. Figure 1. Open in new tabDownload slide Establishment of a clonal cell line (rBM25/S3) from the bone marrow of an adult female rat. Isolation process of rBM25/S3 cells. Primary culture at day 66 (just before the first subcultivation) showing cells with lipid droplets in the cytoplasm (A-1); small asteroid rBM25/S3 cells established by a limiting dilution method at day 192 (PDL 117) (A-2). Bars indicate 50 μm (A). rBM25/S3 cells rapidly grew with a doubling time of 24 hours for at least 300 PDLs over a period of 300 days on fibronectin-coated dishes in the presence of EGF (10 ng/ml) and PDGF-BB (10 ng/ml). Arrow indicates the timing of the single cell cloning (B). Indispensability of EGF and PDGF-BB for the growth of the cells (C). Abbreviations: EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PDL, population doubling level. The rBM25/S3 cells were small and asteroid in shape (Fig. 1A-2). When plated at 2.5 × 103 cells per cm2 on fibronectin substratum with EGF and PDGF-BB, the cells grew with a doubling time of ∼24 hours without any appreciable change in phenotypes until at least 300 PDLs over a period of 300 days (Fig. 1B). Both EGF and PDGF-BB were essential for their growth, the growth rate being reduced to 87%, 48%, and 9% of the control level in media lacking EGF, PDGF-BB, and both factors, respectively (Fig. 1C). Figure 2. Open in new tabDownload slide Karyotypic analysis of rBM25/S3 cells. Normal chromosome distribution in rBM25/S3 cells at least until PDL 307 (A). A karyotype of rBM25/S3 cells at 253 PDL showing a normal diploid pattern of female rat origin (40XX) (B). Abbreviation: PDL, population doubling level. Figure 2. Open in new tabDownload slide Karyotypic analysis of rBM25/S3 cells. Normal chromosome distribution in rBM25/S3 cells at least until PDL 307 (A). A karyotype of rBM25/S3 cells at 253 PDL showing a normal diploid pattern of female rat origin (40XX) (B). Abbreviation: PDL, population doubling level. rBM25/S3 cells stably retained the original normal diploid karyotype (40XX) of female rats up to 307 PDL (Fig. 2). FACS analysis showed that the cells were strongly positive for CD29 and CD90, weakly positive for CD44 and CD49b, and negative for CD45 (Fig. 3). The cells were positively stained for vimentin and fibronectin (Fig. 4), well-known mesenchymal marker proteins [26, 27]. These results indicate that rBM25/S3 cells are of mesenchymal, not hematopoietic, cell origin. Figure 3. Open in new tabDownload slide Fluorescence-activated cell sorting analysis of marker protein expression in rBM25/S3 cells at 220–230 population doubling levels. Peaks in white, stained with specific antibodies; peaks in gray, obtained with isotype control antibodies. Figure 3. Open in new tabDownload slide Fluorescence-activated cell sorting analysis of marker protein expression in rBM25/S3 cells at 220–230 population doubling levels. Peaks in white, stained with specific antibodies; peaks in gray, obtained with isotype control antibodies. Figure 4. Open in new tabDownload slide Immunostaining of vimentin and fibronectin in rBM25/S3 cells. The cells were stained with tetramethylrhodamine isothiocyanate-labeled antibody against vimentin or fibronectin (in red). Nuclei were stained with 4,6-diamidino-2-phenylindole (in blue). Bars indicate 50 μm. Figure 4. Open in new tabDownload slide Immunostaining of vimentin and fibronectin in rBM25/S3 cells. The cells were stained with tetramethylrhodamine isothiocyanate-labeled antibody against vimentin or fibronectin (in red). Nuclei were stained with 4,6-diamidino-2-phenylindole (in blue). Bars indicate 50 μm. We isolated another clone (rBM25/S6) from the same mother culture rBM25 by the limiting dilution method. Furthermore, we newly established a clonal cell line, rBM48/F8–1, from bone marrow cells of a GFP-transgenic Wistar male rat under similar conditions except for earlier first subcultivation at day 29. These cells have characteristics similar to those of rBM25/S3 cells (supplemental online Fig. 1A-1E). These results indicate that the present culture conditions, although incidentally found and empirical, are useful for reproducible isolation of the stem/progenitor cells of mesenchymal origin. Differentiation of Bone Marrow Cell Lines into Hepatocyte-Like Cells in Culture The bone marrow cell lines (rBM25/S3, rBM25/S6, and rBM48/F8–1) were induced to differentiate into hepatocytes under conditions described previously [11], that is, on Matrigel substratum with HGF and FGF-4. After 7-day culture, morphology of these cells became similar to that of hepatocytes, with a large round nucleus and dark cytoplasm with many granules, except for small lipid droplets in the cytoplasm (Fig. 5A and supplemental online Fig. 1F). By RT-PCR, mRNAs of hepatocyte-specific genes such as albumin, G6Pase, TO, and TAT were detected in all the cultures (Fig. 5B). CYP1A1 and -1A2 mRNAs were also detected in the cultures of rBM25/S3 and rBM48/F8–1 cells but not in rBM25/S6 cells (Fig. 5B). Western blot analysis revealed that differentiated rBM25/S3 cells, but not undifferentiated rBM25/S3 cells, produced hepatocyte-specific proteins such as albumin, HNF1α, and HNF4α (Fig. 5C). Immunostaining using the same antibodies resulted in detection of albumin and HNF4α in rBM25/S3-derived hepatocyte-like cells (Fig. 5D). As expected, albumin and HNF4α were mainly localized in the cytoplasm and nuclei, respectively. In addition to these hepatic functions, the rBM25/S3-derived hepatocyte-like cells also expressed genes related to adipogenesis, such as PPARγ, FAS, LPL, ALBP, adipsin, and resistin, and accumulated lipid droplets in the cytoplasm (supplemental online Fig. 2). The clonal cell line rBM25/S3 could also differentiate into osteocyte-like cells showing alkaline phosphatase activity under distinct culture conditions for the induction of differentiation (supplemental online Fig. 3). Figure 5. Open in new tabDownload slide Differentiation of rBM25/S3, rBM25/S6, and rBM48/F8–1 cells into hepatocyte-like cells in vitro. Morphology of rBM25/S3 cells cultured for 7 days on Matrigel with hepatocyte growth factor (HGF) (20 ng/ml) and fibroblast growth factor (FGF)-4 (10 ng/ml). Bar indicates 100 μm (A). Reverse transcription-PCR for hepatocyte-specific mRNAs. Lanes 2, 4, and 6: rBM25/S3, rBM25/S6, and rBM48/F8–1 cells cultured on fibronectin with EGF and platelet-derived growth factor-BB (conditions for cell propagation), respectively. Lanes 3, 5, and 7: rBM25/S3, rBM25/S6, and rBM48/F8–1 cells cultured on Matrigel with HGF and FGF-4 (differentiation-inducing conditions) for 7 days, respectively. Water (lane 1) or adult rat liver (lane 8) was used as a negative or positive control (B). Western blot analysis for albumin, HNF1α, HNF4α, and GFP in the differentiated and undifferentiated rBM25/S3/GFP cells. HepG2 cells served as a positive control for expression of the hepatocyte-specific proteins. β-Actin served as an internal control (C). rBM25/S3 cells before and after the induction of differentiation into hepatocyte-like cells were stained with specific antibodies against albumin and HNF4α. A human hepatoblastoma cell line, HepG2, was used as a positive control. Differentiated rBM25/S3 cells treated with an isotype IgG is shown as a negative control. Nuclei were stained with 4,6-diamidino-2-phenylindole (insets). Bars indicate 100 μm unless otherwise indicated (D). Abbreviations: CYP, cytochrome P450; Diff., differentiated; G6Pase, glucose 6-phosphatase; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; GFP, green fluorescent protein; HNF, hepatocyte nuclear factor; N, nested; PCR, polymerase chain reaction; TAT, tyrosine aminotransferase; TO, triptophan-2,3-dioxygenase; Undiff., undifferentiated. Figure 5. Open in new tabDownload slide Differentiation of rBM25/S3, rBM25/S6, and rBM48/F8–1 cells into hepatocyte-like cells in vitro. Morphology of rBM25/S3 cells cultured for 7 days on Matrigel with hepatocyte growth factor (HGF) (20 ng/ml) and fibroblast growth factor (FGF)-4 (10 ng/ml). Bar indicates 100 μm (A). Reverse transcription-PCR for hepatocyte-specific mRNAs. Lanes 2, 4, and 6: rBM25/S3, rBM25/S6, and rBM48/F8–1 cells cultured on fibronectin with EGF and platelet-derived growth factor-BB (conditions for cell propagation), respectively. Lanes 3, 5, and 7: rBM25/S3, rBM25/S6, and rBM48/F8–1 cells cultured on Matrigel with HGF and FGF-4 (differentiation-inducing conditions) for 7 days, respectively. Water (lane 1) or adult rat liver (lane 8) was used as a negative or positive control (B). Western blot analysis for albumin, HNF1α, HNF4α, and GFP in the differentiated and undifferentiated rBM25/S3/GFP cells. HepG2 cells served as a positive control for expression of the hepatocyte-specific proteins. β-Actin served as an internal control (C). rBM25/S3 cells before and after the induction of differentiation into hepatocyte-like cells were stained with specific antibodies against albumin and HNF4α. A human hepatoblastoma cell line, HepG2, was used as a positive control. Differentiated rBM25/S3 cells treated with an isotype IgG is shown as a negative control. Nuclei were stained with 4,6-diamidino-2-phenylindole (insets). Bars indicate 100 μm unless otherwise indicated (D). Abbreviations: CYP, cytochrome P450; Diff., differentiated; G6Pase, glucose 6-phosphatase; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; GFP, green fluorescent protein; HNF, hepatocyte nuclear factor; N, nested; PCR, polymerase chain reaction; TAT, tyrosine aminotransferase; TO, triptophan-2,3-dioxygenase; Undiff., undifferentiated. Rescue of 90%-Hepatectomized Rats with Acute Liver Failure by Transplantation of Hepatocyte-Like Cells Derived from rBM25/S3/GFP Cells We examined whether the hepatocyte-like cells derived from rat bone marrow show any significant physiological function in vivo (Fig. 6A). rBM25/S3 cells were used since they showed a gene expression profile most similar to that of normal hepatocytes among the cell lines established. The cells were stably transformed with pEGFP-N2 and induced to differentiate into hepatocyte-like cells under the conditions described above. Figure 6. Open in new tabDownload slide Rescue of rats in acute liver failure due to 90% hepatectomy by transplantation of differentiated rBM25/S3/GFP cells. A scheme of the experimental procedure (A). Survival rates in the 90%-hepatectomized rats with or without cell transplantation. The rats underwent 90% hepatectomy 7 days after intrasplenic transplantation of undifferentiated mother cells (circles, 7 rats) or differentiated hepatocyte-like cells (squares, 15 rats). Control rats underwent only 90% hepatectomy (crosses, 13 rats) (B). The regenerated liver (C-1) and spleen (C-2) in a rat at 37 days after transplantation of differentiated rBM25/S3 cells. Liver weight (10.1 g) and blood ammonia level (121 μg/dl) of the rat indicate nearly complete regeneration of the liver. The liver (C-3) and spleen (C-4) of a normal rat at the same age are shown for comparison. Abbreviations: CTx, cell transplantation; wk, week. Figure 6. Open in new tabDownload slide Rescue of rats in acute liver failure due to 90% hepatectomy by transplantation of differentiated rBM25/S3/GFP cells. A scheme of the experimental procedure (A). Survival rates in the 90%-hepatectomized rats with or without cell transplantation. The rats underwent 90% hepatectomy 7 days after intrasplenic transplantation of undifferentiated mother cells (circles, 7 rats) or differentiated hepatocyte-like cells (squares, 15 rats). Control rats underwent only 90% hepatectomy (crosses, 13 rats) (B). The regenerated liver (C-1) and spleen (C-2) in a rat at 37 days after transplantation of differentiated rBM25/S3 cells. Liver weight (10.1 g) and blood ammonia level (121 μg/dl) of the rat indicate nearly complete regeneration of the liver. The liver (C-3) and spleen (C-4) of a normal rat at the same age are shown for comparison. Abbreviations: CTx, cell transplantation; wk, week. A rat model of acute liver failure was made by 90% hepatectomy, in which all of the animals died within 48 hours with blood ammonia levels increased by 25∼35 fold (Fig. 6B; Table 1). Intrasplenic transplantation of rBM25/S3/GFP-derived hepatocyte-like cells (7 × 107 cells per rat) preceding 90% hepatectomy by 7 days prolonged the survival periods of the recipient rats and even rescued 5 of the 15 animals (Fig. 6B). The surviving rats showed a transient increase in blood ammonia level by threefold at 2 days after 90% hepatectomy, and the level gradually returned to the control level in 3∼4 weeks (Table 1). The cell transplantation 1 day before or immediately after 90% hepatectomy failed to support survival of the recipient rats (data not shown). Undifferentiated rBM25/S3 cells, even when transplanted 7 days prior to 90% hepatectomy, could not affect fatality of the operation, that is, all the rats died within 48 hours with severe increase in blood ammonia (Fig. 6B; Table 1). Our control experiments showed that blood ammonia level increased after death by ∼350 μg/dl at most, and we neglected the samples taken after more than 1 hour after death. The liver of the surviving rats regenerated to the original size within 4 weeks after 90% hepatectomy (Fig. 6C). Table 1. Blood ammonia levels in the 90%-hepatectomized rats Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Open in new tab Table 1. Blood ammonia levels in the 90%-hepatectomized rats Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Open in new tab Double immunostaining showed expression of albumin in the transplanted GFP-positive cells that were present as clusters in the livers as well as in the spleens of recipient rats 7 days after transplantation (Fig. 7A). Oil red O staining also showed the clusters of the transplanted cells both in the livers and the spleens of recipient rats (supplemental online Fig. 4). Genomic PCR and Western blot analysis for GFP showed a gradual increase in the relative amounts of transplanted cells in the livers and spleens of recipient rats (Figs. 7B, 7C). Furthermore, Western blotting indicated the presence of a considerable amount of the transplanted cells in the recipient liver even at 101 days after transplantation (Fig. 7C). These results indicate that the intrasplenically transplanted cells were partially recruited from the spleen to engraft in the liver. The hepatocyte-like cells induced from rBM25/S3 cells consistently expressed CXCR4, a chemokine receptor for SDF-1, which is expressed in the liver (Fig. 7D) [28]. In addition, the cells expressed hepatotrophic factors (HGF and TGF-α) and their receptors (c-Met and EGFR) (Fig. 7E), indicating possible involvement of these molecules in the chemotactic recruitment and autocrine/paracrine growth stimulation. Figure 7. Open in new tabDownload slide Presence of transplanted rBM25/S3 cells in the spleen and liver of recipient rats before and after 90% hepatectomy. Detection of intrasplenically transplanted rBM25/S3 cells by double immunostaining for GFP (in red) and albumin (in green) in the spleen and liver tissues 7 days after the CTx. The tissues were successively treated with rabbit anti-human albumin antibody, mouse anti-GFP antibody, Alexa Fluor 488-labeled goat anti-rabbit IgG (green), and Alexa Fluor 594-labeled goat anti-mouse IgG (red). Nuclei were stained with DAPI (in blue). Upper and lower bars indicate 100 μm and 50 μm, respectively (A). Genomic polymerase chain reaction of GFP gene of spleen and liver cells. The samples were obtained from the recipient rats 1, 9, and 11 days after CTx, that is, 6 days before hepatectomy and 2 and 4 days after hepatectomy, respectively (B). Western blot analysis for GFP protein in spleen and liver cells. Protein samples were obtained from the recipient rats 1, 7, 9, 11, 13, and 101 days after CTx, that is, 6 days before hepatectomy and 0, 2, 4, 6, 9, and 94 days after hepatectomy, respectively (C). Reverse transcription-polymerase chain reaction (RT-PCR) for a chemokine, SDF-1, and its receptor, CXCR4, mRNAs. Lane 2, fresh rat bone marrow cells; lane 3, rBM25/S3 cells cultured on fibronectin with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-BB (the conditions for cell propagation); lanes 4 and 5, rBM25/S3 cells cultured on Matrigel with HGF and fibroblast growth factor (FGF)-4 (the differentiation-inducing conditions) for 4 and 7 days, respectively. Water (lane 1) or adult rat liver (lane 6) was used as a negative or positive control (D). RT-PCR for trophic factors (tumor necrosis factor-α, HGF, and TGFα) and their receptors (c-Met and EGFR) mRNAs. Lane 2, fresh rat bone marrow cells; lane 3, rBM25/S3 cells cultured on fibronectin with EGF and PDGF-BB (the conditions for cell propagation); lane 4, rBM25/S3 cells cultured on Matrigel with HGF and FGF-4 (the differentiation-inducing conditions) for 7 days; lane 5, adult rat liver. Water (lane 1) was used as a negative control (E). Abbreviations: CBB, Coomassie Brilliant Blue; CTx, cell transplantation; CXCR, chemokine (C-X-C motif) receptor 4; DAPI, 4,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; GFP, green fluorescent protein; HGF, hepatocyte growth factor; NS, normal rat spleen cells; S3, unmarked rBM25/S3 cells differentiated into hepatocyte-like cells; S3/GFP, GFP-marked rBM25/S3 cells differentiated into hepatocyte-like cells; SDF, stromal cell-derived factor-1; TGF, transforming growth factor-α. Figure 7. Open in new tabDownload slide Presence of transplanted rBM25/S3 cells in the spleen and liver of recipient rats before and after 90% hepatectomy. Detection of intrasplenically transplanted rBM25/S3 cells by double immunostaining for GFP (in red) and albumin (in green) in the spleen and liver tissues 7 days after the CTx. The tissues were successively treated with rabbit anti-human albumin antibody, mouse anti-GFP antibody, Alexa Fluor 488-labeled goat anti-rabbit IgG (green), and Alexa Fluor 594-labeled goat anti-mouse IgG (red). Nuclei were stained with DAPI (in blue). Upper and lower bars indicate 100 μm and 50 μm, respectively (A). Genomic polymerase chain reaction of GFP gene of spleen and liver cells. The samples were obtained from the recipient rats 1, 9, and 11 days after CTx, that is, 6 days before hepatectomy and 2 and 4 days after hepatectomy, respectively (B). Western blot analysis for GFP protein in spleen and liver cells. Protein samples were obtained from the recipient rats 1, 7, 9, 11, 13, and 101 days after CTx, that is, 6 days before hepatectomy and 0, 2, 4, 6, 9, and 94 days after hepatectomy, respectively (C). Reverse transcription-polymerase chain reaction (RT-PCR) for a chemokine, SDF-1, and its receptor, CXCR4, mRNAs. Lane 2, fresh rat bone marrow cells; lane 3, rBM25/S3 cells cultured on fibronectin with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-BB (the conditions for cell propagation); lanes 4 and 5, rBM25/S3 cells cultured on Matrigel with HGF and fibroblast growth factor (FGF)-4 (the differentiation-inducing conditions) for 4 and 7 days, respectively. Water (lane 1) or adult rat liver (lane 6) was used as a negative or positive control (D). RT-PCR for trophic factors (tumor necrosis factor-α, HGF, and TGFα) and their receptors (c-Met and EGFR) mRNAs. Lane 2, fresh rat bone marrow cells; lane 3, rBM25/S3 cells cultured on fibronectin with EGF and PDGF-BB (the conditions for cell propagation); lane 4, rBM25/S3 cells cultured on Matrigel with HGF and FGF-4 (the differentiation-inducing conditions) for 7 days; lane 5, adult rat liver. Water (lane 1) was used as a negative control (E). Abbreviations: CBB, Coomassie Brilliant Blue; CTx, cell transplantation; CXCR, chemokine (C-X-C motif) receptor 4; DAPI, 4,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; GFP, green fluorescent protein; HGF, hepatocyte growth factor; NS, normal rat spleen cells; S3, unmarked rBM25/S3 cells differentiated into hepatocyte-like cells; S3/GFP, GFP-marked rBM25/S3 cells differentiated into hepatocyte-like cells; SDF, stromal cell-derived factor-1; TGF, transforming growth factor-α. Discussion Stem cell-based therapy is a promising strategy for treating fatal hepatic failure considering the serious shortage of donor livers. Availability of highly functional cells in a large quantity is a prerequisite for successful cell therapy. In the present study, we established three clonal stem cell lines from the bone marrow of adult rats. rBM25/S3, a representative cell line, showed the following properties: (a) the cells grew rapidly with a doubling time of approximately 24 hours at least until PDL 300; (b) they stably retained cellular phenotypes including the diploid karyotype; (c) they showed multipotency as demonstrated by differentiation into osteocytes as well as hepatocytes; (d) they differentiated into albumin- and HNF4α-positive hepatocyte-like cells as shown by immunostaining; (e) they differentiated into hepatocyte-like cells within 1 week. We had thought that these properties made rBM25/S3 cells an ideal cell source for transplantation into an animal model of fatal liver failure, and indeed intrasplenic transplantation of hepatocyte-like cells derived from rBM25/S3/GFP cells efficiently rescued 90%-hepatectomized rats, all of which otherwise died within 48 hours. rBM25/S3 cells are strongly positive for CD29, CD90, vimentin, and fibronectin, weakly positive for CD44 and CD49b, and negative for CD45, indicating the mesenchymal origin. Typical MSCs have been reported to be strongly positive for CD29, CD44, and CD90 [7], and MAPCs have been reported to be weakly positive for CD49b and CD90 and negative for CD44 [29]. rBM25/S3 cells are, therefore, distinct from MSCs or MAPCs, although the three types of cells share some cell surface markers. At present, it is not clear whether the cell types are at different differentiation stages of a common lineage or whether they originate from distinct cell lineages. rBM25/S3/GFP-derived hepatocyte-like cells were detected by double immunostaining for GFP and albumin and oil red O staining in the spleen and liver 7 days after intrasplenic transplantation (Fig. 7A; supplemental online Fig. 4), indicating that the transplanted cells were not only trapped in the spleen but also efficiently recruited into the liver. RT-PCR analysis demonstrated that CXCR4, a chemokine receptor for SDF-1 [28], was induced in rBM25/S3/GFP cells during cultivation under the conditions for hepatic differentiation (Fig. 7D). The induction of CXCR4 was probably due to HGF contained in the medium, since HGF has been shown to upregulate CXCR4 in human hematopoietic stem cells [30]. Transcripts of SDF-1, the ligand of CXCR4, were detected in liver cells (Fig. 7D). These results indicate that the intrasplenically transplanted cells were recruited into liver not only by a hemodynamic process but also by an active chemotaxic mechanism. In the present study, engraftment of rBM25/S3-derived hepatocyte-like cells was confirmed by Western blot analysis for GFP protein in the liver of syngeneic Sprague-Dawley recipient rats 101 days after intrasplenic transplantation without immunosuppression (Fig. 7C). We and other groups observed that intrasplenically transplanted hepatocytes from Donryu and Fischer 344 rats survived in the spleen of syngeneic recipient rats for at least 11 and 15 months after transplantation, respectively, without immunosuppressants [31, 32]. Thus, acquisition of immunologic tolerance may be relatively easy in the cell transplantation system among syngeneic rats, although the mechanism still remains unclear. In 7-day culture under the differentiation conditions with Matrigel, HGF, and FGF-4, rBM25/S3 cells were induced to express adipogenic genes such as PPARγ, FAS, LPL, ALBP, adipsin, and resistin as well as the hepatic genes albumin, G6Pase, TO, TAT, CYP1A1, CYP1A2, HNF1α, and HNF4α (Fig. 5B–5D; supplemental online Fig. 2A, 2B). After transplantation, nodules of transplanted rBM25/S3 cells were identified as lipid-positive cell populations in the liver (supplemental online Fig. 4). Accumulation of lipid droplets in hepatocytes is a feature of fatty liver. Shteyer et al. [33] reported that hepatocytes transiently accumulate lipid during early liver regeneration after partial hepatectomy in mice, and the hepatocellular lipid accumulation is essential for normal liver regeneration. We also observed transient lipid accumulation in the regenerating liver after 90% hepatectomy, which returned to the normal level by day 27 (data not shown). At present, it is not clear whether the expression of adipogenic genes of rBM25/S3 cells reflects intermediate property of the cells between hepatocytes and adipocytes or is related to lipid metabolism observed in hepatocytes. Wada et al. [34] showed that multipotent stem cells express different gene sets at an early stage of differentiation that are specific to different cell lineages, and with the progression of differentiation the gene expression profile becomes progressively restricted to a specific cell type. HSCs can differentiate into cells with mature hepatic phenotypes in vivo [9] as well as in vitro [15–17]. Although transplantation of HSCs was reported to rescue mice with a specific type of chronic fatal liver failure due to deficiency of fumarylacetoacetate hydrolase [35], the curative effect of HSCs for liver failure in general remains controversial [36]. In an animal model of acute liver failure, transplantation of hepatocyte-like cells induced from bone marrow cells in culture showed no remarkable effects (data not shown). Dahlke et al. [37] also reported marginal effects of whole bone marrow cells on acute liver failure induced by carbon tetrachloride and retrorsine in rats. The unsuccessful therapeutic trials were probably due to limited growth and differentiation capacity of stem cells contained in the bone marrow. On the other hand, embryonic stem (ES) cells have high proliferation capacity and are in principle capable of forming all adult tissues. However, it has been reported that transplantation of ES-derived cells resulted in the formation of teratomas. Although elimination of undifferentiated cells from the cell population may suppress tumor formation [38, 39], the problem remains as a serious challenge against possible utility of ES cells for cell therapy. The properties of rBM25/S3 cells demonstrated in the present study (extremely high proliferation capacity, efficient differentiation into hepatocytes in vitro, and no tumor formation over a 3-month observation period even after transplantation of a large number of cells) indicate the possibility of overcoming such problems associated with future therapeutic application of bone marrow-derived stem cells. In experimental settings similar to the present study, Aoki et al. [40] showed that intrasplenic transplantation of encapsulated primary hepatocytes (2 × 107 cells per rat) rescued 90%-hepatectomized rats at a rate of 9/14 (64%). In their case, however, even control rats uninjected or injected with capsule alone survived at rates of 2/14 (14%) and 3/14 (21%), respectively. We performed the present experiments under more strict conditions, that is, all the hepatectomized rats died within 48 hours unless transplanted with appropriate cells. Intrasplenic transplantation of rBM25/S3/GFP-derived hepatocyte-like cells rescued 5 rats among the 15 fatally hepatectomized rats (33%) (Fig. 6B). After transplantation, the transplanted cells apparently engrafted and propagated in the liver of recipient rats as indicated by genomic PCR and Western blot analysis for GFP (Fig. 7B, 7C). However, we could not unequivocally prove that the transplanted cells fully differentiated into cells with functions comparable to endogenous hepatocytes. These issues and the possibility of cell fusion between the transplanted cells and endogenous hepatocytes remain to be solved. Conclusion We established several clonal stem cell lines that gave rise to hepatocyte-like cells in a large quantity in culture and demonstrated that transplantation of the cells rescued rats with fatal liver failure induced by 90% hepatectomy. 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Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2007 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Isolation of a Bone Marrow-Derived Stem Cell Line with High Proliferation Potential and Its Application for Preventing Acute Fatal Liver Failure
JF - Stem Cells
DO - 10.1634/stemcells.2007-0078
DA - 2007-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/isolation-of-a-bone-marrow-derived-stem-cell-line-with-high-qKw3290grI
SP - 2855
EP - 2863
VL - 25
IS - 11
DP - DeepDyve
ER -