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Hepatocyte Growth Factor Induces Differentiation of Adult Rat Bone Marrow Cells into a Hepatocyte Lineage in Vitro
Biochemical and Biophysical Research Communications 279, 500 –504 (2000) doi:10.1006/bbrc.2000.3985, available online at http://www.idealibrary.com on
Hepatocyte Growth Factor Induces Differentiation of Adult Rat Bone Marrow Cells into a Hepatocyte Lineage in Vitro Seh-Hoon Oh,* Masahiro Miyazaki,* ,1 Hirosuke Kouchi,* Yusuke Inoue,* Masakiyo Sakaguchi,* Toshiya Tsuji,* Nobuyuki Shima,† Kanji Higashio,† and Masayoshi Namba* *Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan; and †Research Institute of Life Science, Snow Brand Milk Products Company, Ltd., Tochigi 329-0512, Japan
Received November 14, 2000
Bone marrow (BM) cells originally include alphafetoprotein (AFP)- and c-Met [a receptor for hepatocyte growth factor (HGF)]-expressing cells. In vitro treatment of BM cells with HGF induced albuminexpressing hepatocyte-like cells. Furthermore, those hepatocyte-like cells expressed cytokeratins 8 and 18, which are typically expressed in normal adult hepatocytes. These findings demonstrate that BM cells include AFP-expressing hepatic progenitor cells that can be differentiated into hepatocytes by HGF in culture, indicating that such cultures are useful resources for cell transplantation therapy for liver diseases. © 2000 Academic Press Key Words: bone marrow cells; hepatocyte growth factor; differentiation; hepatocyte lineage.
Stem cells are thought to be pluripotent cells that can be differentiated into a variety of cell or tissue types and can be ideal resources of transplantation therapy. In recent studies, bone marrow (BM) cells developed into hepatocytes by in vivo transplantation (1– 4). These findings indicate that BM cells include pluripotent stem cells, but the mechanism of BM cell differentiation into a hepatocyte lineage is still not clear. The cell differentiation and regeneration are controlled by growth factor(s) or cytokine(s). Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, shows mitogenic, motogenic and morphogenic activities for a wide variety of cells that express the HGF receptor c-Met, a transmembrane protein possessing an intracellular tyrosine kinase domain (5, 6). Moreover, HGF plays an To whom correspondence should be addressed. Fax: ⫹81-86-2357400. E-mail: [email protected]. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
essential role in the development and regeneration of the liver (7–12). In the present study, to elucidate the mechanism of differentiation of BM cells into hepatocytes, we examined cytological effects of HGF on adult rat BM cells in culture. As a result, we found that HGF efficiently induced differentiation of BM cells into albumin-expressing hepatocyte-like cells at high concentrations of 0.5 to 5 g/ml in culture. MATERIALS AND METHODS Cell cultures. BM cells were collected from the femora of Wistar rats (2-months-old or more). The marrow cells were precultured in a mixture (1:1) of Dulbecco’s modified Eagle medium and Ham’s medium F12 (DF medium) supplemented with 10% fetal bovine serum (FBS). After 60 min of incubation, nonadherent cells were collected and washed with fresh serum-free DF medium. The cells were reinoculated in the serum-free DF medium at a cell density of 1 ⫻ 10 4/cm 2 in the presence or absence of HGF, which was the ⌬5 variant, the mature two-chain form, and was produced recombinantly and purified as described previously (13). The detailed culture conditions are shown in Fig. 1a. The cells were cultured for 21 days. HGF was freshly added with medium change every 3 days. Detection of albumin, alpha-fetoprotein (AFP), and c-Met mRNAs. Total RNA was isolated from the adult rat liver and BM cells treated with or without HGF by the guanidinium thiocyanatephenol method (Fig. 1a), and 1 g RNA was used for cDNA synthesis. The resulting RT products were amplified under the following conditions: at 94°C for 4 min followed by 30 cycles at 94°C for 30 s, 62°C for 90 s (for albumin) or 55°C for 45 s (for AFP, 1st PCR) or 60°C for 45 s (for AFP, 2nd PCR) or 58°C for 1 min [for c-Met and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] and 72°C for 1 min, and then a final cycle at 72°C for 4 min. The albumin primers used were 5⬘-ATACACCCAGAAAGCACCTC-3⬘ (sense strand) and 5⬘-CACGAATTGTGCGAATGTCAC-3⬘ (antisense strand), which delineated a 436-bp product. The AFP primers used were 5⬘-AACAGCAGAGTGCTGCAAAC-3⬘ (sense strand) and 5⬘-AGGTTTCGTCCCTCAGAAAG-3⬘ (antisense strand), which delineated a 686-bp product. The nested primers 5⬘CACCATCGAGCTCGCCTATT-3⬘ and5⬘-TGATGCAGAGCCTCCTGTTG-3⬘ delineated a 622-bp product. The c-Met primers used were 5⬘-CAGTGATGATCTCAATGGGCAAT-3⬘ (sense strand) and 5⬘-AATGCCCTCTTCCTATGACTTC-3⬘ (antisense strand), which
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delineated a 725-bp product. The GAPDH primers used were 5⬘-ATCACTGCCACTCAGAAGAC-3⬘ (sense strand) and 5⬘-TGAGGGAGATGCTCAGTGTT-3⬘ (antisense strand), which delineated a 580-bp product. The amplified products were subjected to electrophoresis in 1% agarose gels and stained with ethidium bromide. The purified PCR products were directly sequenced using an AmpliTaq cycle sequencing kit (Perkin-Elmer Setus, Branchburg, NJ). Immunocytochemistry. For immunocytochemistry, cells were cultured under the same conditions as above except for the plating of cells on coverslips (24 ⫻ 24 mm) coated with 0.3% type I collagen, which was extracted from the rat tail tendon by the method of Michalopoulos and Pitot (14). After 21 days of culture, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 30 min and then treated with 1% bovine serum albumin, 0.1% Triton ⫻ 100, and 0.05% sodium azide in PBS (a blocking solution) at 4°C for 30 min. Cells were then reacted at 4°C overnight with primary antibodies, such as anti-rat albumin (Dako Japan, Kyoto), anti-rat AFP (Funacoshi, Tokyo), anti-rat cytokeratin (CK) 8 (Funacoshi) and anti-rat CK 18 (Funacoshi), which had been diluted at 1:100 with the blocking solution. After washing with PBS, cells were incubated at 4°C for 3 h with a second antibody, TRITCconjugated anti-rat IgG rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100 dilution with the blocking solution), and with 1 mM Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) for nuclear staining. The cells were then observed under a fluorescent microscope.
RESULTS AND DISCUSSION BM cell cultures were treated with or without HGF at concentrations of 5 ng/ml to 5 g/ml for the first 5 days of culture and then maintained for further 16 days in the presence or absence of HGF at 5 ng/ml. Microscopic observation of hepatocyte-like cells in BM cell cultures treated with HGF at high concentrations of 0.5 to 5 g/ml prompted us to analyze theses cultures for genetical and biochemical evidence of differentiation into hepatocytes. In the following experiments, we treated BM cell cultures with HGF at 1 g/ml for the initial 5 days of culture, as shown in Fig. 1a. We assessed the differentiation of BM cells into hepatocyte-like cells by HGF in vitro by the following three approaches: (i) determination of the morphologic effects of HGF on BM cell cultures; (ii) gene expressions of albumin and AFP in BM cell cultures; and (iii) cytochemical staining of hepatocyte markers such as albumin, AFP, and CKs 8 and 18 (Fig. 1a). We first examined whether BM cells express the c-met gene encoding the HGF receptor. c-Met mRNA was detected by RT-PCR in freshly isolated BM cells as well as in normal rat liver as a positive control (Fig. 1b, lanes 1 and 2). These results indicate that adult BM cells can respond to HGF. Liver gene expression during ontogeny is characterized by on-off switches, e.g., between serum AFP and albumin (15). Soon after birth, AFP expression in the liver decreases to a very low level, in parallel with a marked reduction of AFP mRNA (16). In addition, production of AFP is associated with normal hepatocyte division (17). Albumin, the most abundant protein syn-
FIG. 1. Culture conditions of BM cells and genetical characterization of the cultured cells. (a) BM cells were cultured in a serumfree medium for 5 days in the presence or absence of a high concentration HGF (1 g/ml). The cells were then cultured in 10% FBScontaining medium in the presence or absence of a low concentration HGF (5 ng/ml). (b) RT-PCR analysis of expression of c-Met, AFP and albumin mRNAs in adult rat liver, BM cells and their cultures.
thesized by hepatocytes, is first expressed in the fetal rat liver on day 11.5 of the development process, and its expression remains in the adult liver (18). To determine whether or not hepatocyte-like cells are originally present in the BM, we tested total RNA extracted from freshly isolated BM cells by RT-PCR for expression of AFP and/or albumin mRNAs. We detected AFP mRNA but not albumin mRNA in freshly isolated BM cells (Fig. 1b, lane 2). Next, we examined the expression of these genes in BM cell cultures treated with or without HGF. Albumin, AFP, c-Met and GAPDH mRNAs were detected by RT-PCR in BM cells grown in the presence of HGF (1 g/ml) during the initial 5 days of culture (Fig. 1b, lanes 4 and 5). These messages, other than GAPDH, were, however, not detected in BM cells grown in culture without HGF (Fig. 1b, lane 3). Withdrawal of the growth factor after 5 days of culture reduced the expression of albumin mRNA but not the expression of AFP mRNA (Fig. 1b, lanes 4 and 5). Although freshly isolated BM cells expressed AFP mRNA (Fig. 1b, lane 2), the message was not detected in BM cell cultures without HGF treatment (Fig. 1b, lane 3). We then directly sequenced the PCR-amplified products. Their sequences were completely consistent with the reported sequences (19, 20) of the normal rat albumin and AFP genes (data not shown). These re-
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FIG. 2. Immunocytochemical localization of albumin and AFP in BM cells cultured for 3 weeks in the presence of HGF. (a, d) Immunofluorescent staining of BM cells with anti-rat albumin or AFP antibody, respectively. (b, e) Nuclear staining of BM cells with Hoechst 33258. Albumin-positive cells have formed a cluster (a), but AFP-positive cells are scattered (d). Both albumin- and AFP-positive cells display morphological characteristics of hepatocytes having a large round nucleus with a few nucleoli and many granules in the cytoplasm (c, f ). a, b, and c; and d, e, and f are the same fields of the BM cell cultures. Bars indicate 50 m.
sults indicate that HGF is essential for maintaining AFP-expressing cells and inducing expression of albumin mRNA in them in BM cell cultures. To localize albumin and AFP production to a particular cell type, we immunocytochemically evaluated HGF-treated BM cell cultures using anti-albumin and anti-AFP antibodies, which recognize cytoplasmic albumin and AFP typically expressed in hepatocytes. Albumin-positive cells formed colonies or were scattered (Figs. 2a and 2b), whereas AFP-positive cells were only scattered (Figs. 2d and 2e). Both albuminand AFP-positive cells displayed morphological characteristics of hepatocytes having a large round nucleus with a few nucleoli and many granules in the cytoplasm (Figs. 2c and 2f ). Finally, we immunocytochemically analyzed the expression of CKs 8 and 18, which are typically expressed in normal adult hepatocytes. Hepatocyte-like cells in HGF-treated BM cell cultures were positively stained for CK 8 (Figs. 3a and 3b) and CK 18 (Figs. 3c and 3d), whereas there were no cells positive for these cytoker-
atins in the absence of HGF (data not shown). Thus, BM-cell-derived hepatocyte-like cells were characteristic of normal hepatocytes. HGF is a ubiquitous and pluripotent cytokine that shows mitogenic, motogenic and morphogenic activities toward a variety of cells via activation of its receptor, c-Met (5, 6). Indeed, we detected c-Met mRNA by RT-PCR in freshly isolated BM cells and their cultures treated with HGF during the initial 5 days. HGF and its receptor, c-Met, may mediate important steps in organogenesis in vitro and play a crucial role in placenta and fetal hepatic growth development in vivo (21). Furthermore, we demonstrated in the present study that HGF could control differentiation of hepatic progenitor cells originally included in BM cells. Many growth factors and cytokines, most notably HGF, epidermal growth factor, transforming growth factor-␣, interleukin-6, tumor necrosis factor-␣, insulin, and norepinephrine, appear to play important roles in liver regeneration (for review, see 10). Fibroblast growth factors and several families of transcription factors including hepatocyte nuclear factors 1, 3, and 4 have been shown to be important components of liver development and differentiation process (for review, see 22). Thus, in future it should be determined whether these growth factors and cytokines other than HGF also contribute to differentiation of BM cells into a hepatocyte lineage. Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell-cell interactions are imperative for coordinated organ func-
FIG. 3. Immunocytochemical staining of CKs 8 and 18 in BM cells cultured in the presence of HGF. (a, c) Immunofluorescent stainings of BM cells with anti-rat CKs 8 and 18 antibodies, respectively. (b, d) Nuclear staining of BM cells with Hoechst 33258. CK 8and 18-positive cells are scattered. a and b; and c and d are the same fields of the BM cell cultures. Bars indicate 50 m.
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tion. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype (for review, see 23). So, it is interesting to determine whether or not cell-cell interactions improve BM cell differentiation into a hepatocyte lineage. BM stem cells are normally present in adult marrow, pluripotent cells, and can differentiate into a variety of cell types, including cells in bone (24), muscle (25), fat (26), tendon, cartilage (27), or cardiomyocytes (28). In this study, we demonstrated that BM cells also include hepatic progenitor cells that express AFP mRNA and can differentiate into hepatocytes in an HGF-dependent manner. Since we detected HGF mRNA in freshly isolated BM cells (data not shown), it is possible that AFP-expressing hepatic progenitor cells are induced from BM stem cells by paracrine HGF. This raises the question as to why AFP-expressing hepatic progenitor cells do not further differentiate into hepatocytes in the BM in vivo. This process may need some stimuli such as liver damage. In the present study, cell culture may have played a role in stimulation for differentiation of hepatic progenitor cells into hepatocytes. In addition, this process may depend on the HGF concentration, which is known to remarkably increase in the blood following liver damage (6). In any case, hepatic progenitor cells in the BM may be a useful resource for transplantation therapy for liver failure regardless of the major histocompatibility complex, since the patient’s own BM cells can be used for transplantation. Cultivation of BM cells in the presence of HGF has the following advantages: (i) it is possible to prepare a hepatocyte-enriched population from BM cells; (ii) it is possible to functionally improve BM-derived hepatocytes by introduction of important genes; and (iii) it is possible to develop a bioartificial liver device with BMderived hepatocytes. In addition, HGF is also useful for treatment of liver cirrhosis (29) and can induce differentiation of hepatic progenitor cells included in the BM into hepatocytes. Taken together, the combination of HGF and BM cells may be useful for treating liver diseases.
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Hepatocyte Growth Factor Induces Differentiation of Adult Rat Bone Marrow Cells into a Hepatocyte Lineage in Vitro Seh-Hoon Oh,* Masahiro Miyazaki,* ,1 Hirosuke Kouchi,* Yusuke Inoue,* Masakiyo Sakaguchi,* Toshiya Tsuji,* Nobuyuki Shima,† Kanji Higashio,† and Masayoshi Namba* *Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan; and †Research Institute of Life Science, Snow Brand Milk Products Company, Ltd., Tochigi 329-0512, Japan
Received November 14, 2000
Bone marrow (BM) cells originally include alphafetoprotein (AFP)- and c-Met [a receptor for hepatocyte growth factor (HGF)]-expressing cells. In vitro treatment of BM cells with HGF induced albuminexpressing hepatocyte-like cells. Furthermore, those hepatocyte-like cells expressed cytokeratins 8 and 18, which are typically expressed in normal adult hepatocytes. These findings demonstrate that BM cells include AFP-expressing hepatic progenitor cells that can be differentiated into hepatocytes by HGF in culture, indicating that such cultures are useful resources for cell transplantation therapy for liver diseases. © 2000 Academic Press Key Words: bone marrow cells; hepatocyte growth factor; differentiation; hepatocyte lineage.
Stem cells are thought to be pluripotent cells that can be differentiated into a variety of cell or tissue types and can be ideal resources of transplantation therapy. In recent studies, bone marrow (BM) cells developed into hepatocytes by in vivo transplantation (1– 4). These findings indicate that BM cells include pluripotent stem cells, but the mechanism of BM cell differentiation into a hepatocyte lineage is still not clear. The cell differentiation and regeneration are controlled by growth factor(s) or cytokine(s). Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, shows mitogenic, motogenic and morphogenic activities for a wide variety of cells that express the HGF receptor c-Met, a transmembrane protein possessing an intracellular tyrosine kinase domain (5, 6). Moreover, HGF plays an To whom correspondence should be addressed. Fax: ⫹81-86-2357400. E-mail: [email protected]. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
essential role in the development and regeneration of the liver (7–12). In the present study, to elucidate the mechanism of differentiation of BM cells into hepatocytes, we examined cytological effects of HGF on adult rat BM cells in culture. As a result, we found that HGF efficiently induced differentiation of BM cells into albumin-expressing hepatocyte-like cells at high concentrations of 0.5 to 5 g/ml in culture. MATERIALS AND METHODS Cell cultures. BM cells were collected from the femora of Wistar rats (2-months-old or more). The marrow cells were precultured in a mixture (1:1) of Dulbecco’s modified Eagle medium and Ham’s medium F12 (DF medium) supplemented with 10% fetal bovine serum (FBS). After 60 min of incubation, nonadherent cells were collected and washed with fresh serum-free DF medium. The cells were reinoculated in the serum-free DF medium at a cell density of 1 ⫻ 10 4/cm 2 in the presence or absence of HGF, which was the ⌬5 variant, the mature two-chain form, and was produced recombinantly and purified as described previously (13). The detailed culture conditions are shown in Fig. 1a. The cells were cultured for 21 days. HGF was freshly added with medium change every 3 days. Detection of albumin, alpha-fetoprotein (AFP), and c-Met mRNAs. Total RNA was isolated from the adult rat liver and BM cells treated with or without HGF by the guanidinium thiocyanatephenol method (Fig. 1a), and 1 g RNA was used for cDNA synthesis. The resulting RT products were amplified under the following conditions: at 94°C for 4 min followed by 30 cycles at 94°C for 30 s, 62°C for 90 s (for albumin) or 55°C for 45 s (for AFP, 1st PCR) or 60°C for 45 s (for AFP, 2nd PCR) or 58°C for 1 min [for c-Met and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] and 72°C for 1 min, and then a final cycle at 72°C for 4 min. The albumin primers used were 5⬘-ATACACCCAGAAAGCACCTC-3⬘ (sense strand) and 5⬘-CACGAATTGTGCGAATGTCAC-3⬘ (antisense strand), which delineated a 436-bp product. The AFP primers used were 5⬘-AACAGCAGAGTGCTGCAAAC-3⬘ (sense strand) and 5⬘-AGGTTTCGTCCCTCAGAAAG-3⬘ (antisense strand), which delineated a 686-bp product. The nested primers 5⬘CACCATCGAGCTCGCCTATT-3⬘ and5⬘-TGATGCAGAGCCTCCTGTTG-3⬘ delineated a 622-bp product. The c-Met primers used were 5⬘-CAGTGATGATCTCAATGGGCAAT-3⬘ (sense strand) and 5⬘-AATGCCCTCTTCCTATGACTTC-3⬘ (antisense strand), which
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delineated a 725-bp product. The GAPDH primers used were 5⬘-ATCACTGCCACTCAGAAGAC-3⬘ (sense strand) and 5⬘-TGAGGGAGATGCTCAGTGTT-3⬘ (antisense strand), which delineated a 580-bp product. The amplified products were subjected to electrophoresis in 1% agarose gels and stained with ethidium bromide. The purified PCR products were directly sequenced using an AmpliTaq cycle sequencing kit (Perkin-Elmer Setus, Branchburg, NJ). Immunocytochemistry. For immunocytochemistry, cells were cultured under the same conditions as above except for the plating of cells on coverslips (24 ⫻ 24 mm) coated with 0.3% type I collagen, which was extracted from the rat tail tendon by the method of Michalopoulos and Pitot (14). After 21 days of culture, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 30 min and then treated with 1% bovine serum albumin, 0.1% Triton ⫻ 100, and 0.05% sodium azide in PBS (a blocking solution) at 4°C for 30 min. Cells were then reacted at 4°C overnight with primary antibodies, such as anti-rat albumin (Dako Japan, Kyoto), anti-rat AFP (Funacoshi, Tokyo), anti-rat cytokeratin (CK) 8 (Funacoshi) and anti-rat CK 18 (Funacoshi), which had been diluted at 1:100 with the blocking solution. After washing with PBS, cells were incubated at 4°C for 3 h with a second antibody, TRITCconjugated anti-rat IgG rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100 dilution with the blocking solution), and with 1 mM Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) for nuclear staining. The cells were then observed under a fluorescent microscope.
RESULTS AND DISCUSSION BM cell cultures were treated with or without HGF at concentrations of 5 ng/ml to 5 g/ml for the first 5 days of culture and then maintained for further 16 days in the presence or absence of HGF at 5 ng/ml. Microscopic observation of hepatocyte-like cells in BM cell cultures treated with HGF at high concentrations of 0.5 to 5 g/ml prompted us to analyze theses cultures for genetical and biochemical evidence of differentiation into hepatocytes. In the following experiments, we treated BM cell cultures with HGF at 1 g/ml for the initial 5 days of culture, as shown in Fig. 1a. We assessed the differentiation of BM cells into hepatocyte-like cells by HGF in vitro by the following three approaches: (i) determination of the morphologic effects of HGF on BM cell cultures; (ii) gene expressions of albumin and AFP in BM cell cultures; and (iii) cytochemical staining of hepatocyte markers such as albumin, AFP, and CKs 8 and 18 (Fig. 1a). We first examined whether BM cells express the c-met gene encoding the HGF receptor. c-Met mRNA was detected by RT-PCR in freshly isolated BM cells as well as in normal rat liver as a positive control (Fig. 1b, lanes 1 and 2). These results indicate that adult BM cells can respond to HGF. Liver gene expression during ontogeny is characterized by on-off switches, e.g., between serum AFP and albumin (15). Soon after birth, AFP expression in the liver decreases to a very low level, in parallel with a marked reduction of AFP mRNA (16). In addition, production of AFP is associated with normal hepatocyte division (17). Albumin, the most abundant protein syn-
FIG. 1. Culture conditions of BM cells and genetical characterization of the cultured cells. (a) BM cells were cultured in a serumfree medium for 5 days in the presence or absence of a high concentration HGF (1 g/ml). The cells were then cultured in 10% FBScontaining medium in the presence or absence of a low concentration HGF (5 ng/ml). (b) RT-PCR analysis of expression of c-Met, AFP and albumin mRNAs in adult rat liver, BM cells and their cultures.
thesized by hepatocytes, is first expressed in the fetal rat liver on day 11.5 of the development process, and its expression remains in the adult liver (18). To determine whether or not hepatocyte-like cells are originally present in the BM, we tested total RNA extracted from freshly isolated BM cells by RT-PCR for expression of AFP and/or albumin mRNAs. We detected AFP mRNA but not albumin mRNA in freshly isolated BM cells (Fig. 1b, lane 2). Next, we examined the expression of these genes in BM cell cultures treated with or without HGF. Albumin, AFP, c-Met and GAPDH mRNAs were detected by RT-PCR in BM cells grown in the presence of HGF (1 g/ml) during the initial 5 days of culture (Fig. 1b, lanes 4 and 5). These messages, other than GAPDH, were, however, not detected in BM cells grown in culture without HGF (Fig. 1b, lane 3). Withdrawal of the growth factor after 5 days of culture reduced the expression of albumin mRNA but not the expression of AFP mRNA (Fig. 1b, lanes 4 and 5). Although freshly isolated BM cells expressed AFP mRNA (Fig. 1b, lane 2), the message was not detected in BM cell cultures without HGF treatment (Fig. 1b, lane 3). We then directly sequenced the PCR-amplified products. Their sequences were completely consistent with the reported sequences (19, 20) of the normal rat albumin and AFP genes (data not shown). These re-
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FIG. 2. Immunocytochemical localization of albumin and AFP in BM cells cultured for 3 weeks in the presence of HGF. (a, d) Immunofluorescent staining of BM cells with anti-rat albumin or AFP antibody, respectively. (b, e) Nuclear staining of BM cells with Hoechst 33258. Albumin-positive cells have formed a cluster (a), but AFP-positive cells are scattered (d). Both albumin- and AFP-positive cells display morphological characteristics of hepatocytes having a large round nucleus with a few nucleoli and many granules in the cytoplasm (c, f ). a, b, and c; and d, e, and f are the same fields of the BM cell cultures. Bars indicate 50 m.
sults indicate that HGF is essential for maintaining AFP-expressing cells and inducing expression of albumin mRNA in them in BM cell cultures. To localize albumin and AFP production to a particular cell type, we immunocytochemically evaluated HGF-treated BM cell cultures using anti-albumin and anti-AFP antibodies, which recognize cytoplasmic albumin and AFP typically expressed in hepatocytes. Albumin-positive cells formed colonies or were scattered (Figs. 2a and 2b), whereas AFP-positive cells were only scattered (Figs. 2d and 2e). Both albuminand AFP-positive cells displayed morphological characteristics of hepatocytes having a large round nucleus with a few nucleoli and many granules in the cytoplasm (Figs. 2c and 2f ). Finally, we immunocytochemically analyzed the expression of CKs 8 and 18, which are typically expressed in normal adult hepatocytes. Hepatocyte-like cells in HGF-treated BM cell cultures were positively stained for CK 8 (Figs. 3a and 3b) and CK 18 (Figs. 3c and 3d), whereas there were no cells positive for these cytoker-
atins in the absence of HGF (data not shown). Thus, BM-cell-derived hepatocyte-like cells were characteristic of normal hepatocytes. HGF is a ubiquitous and pluripotent cytokine that shows mitogenic, motogenic and morphogenic activities toward a variety of cells via activation of its receptor, c-Met (5, 6). Indeed, we detected c-Met mRNA by RT-PCR in freshly isolated BM cells and their cultures treated with HGF during the initial 5 days. HGF and its receptor, c-Met, may mediate important steps in organogenesis in vitro and play a crucial role in placenta and fetal hepatic growth development in vivo (21). Furthermore, we demonstrated in the present study that HGF could control differentiation of hepatic progenitor cells originally included in BM cells. Many growth factors and cytokines, most notably HGF, epidermal growth factor, transforming growth factor-␣, interleukin-6, tumor necrosis factor-␣, insulin, and norepinephrine, appear to play important roles in liver regeneration (for review, see 10). Fibroblast growth factors and several families of transcription factors including hepatocyte nuclear factors 1, 3, and 4 have been shown to be important components of liver development and differentiation process (for review, see 22). Thus, in future it should be determined whether these growth factors and cytokines other than HGF also contribute to differentiation of BM cells into a hepatocyte lineage. Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell-cell interactions are imperative for coordinated organ func-
FIG. 3. Immunocytochemical staining of CKs 8 and 18 in BM cells cultured in the presence of HGF. (a, c) Immunofluorescent stainings of BM cells with anti-rat CKs 8 and 18 antibodies, respectively. (b, d) Nuclear staining of BM cells with Hoechst 33258. CK 8and 18-positive cells are scattered. a and b; and c and d are the same fields of the BM cell cultures. Bars indicate 50 m.
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tion. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype (for review, see 23). So, it is interesting to determine whether or not cell-cell interactions improve BM cell differentiation into a hepatocyte lineage. BM stem cells are normally present in adult marrow, pluripotent cells, and can differentiate into a variety of cell types, including cells in bone (24), muscle (25), fat (26), tendon, cartilage (27), or cardiomyocytes (28). In this study, we demonstrated that BM cells also include hepatic progenitor cells that express AFP mRNA and can differentiate into hepatocytes in an HGF-dependent manner. Since we detected HGF mRNA in freshly isolated BM cells (data not shown), it is possible that AFP-expressing hepatic progenitor cells are induced from BM stem cells by paracrine HGF. This raises the question as to why AFP-expressing hepatic progenitor cells do not further differentiate into hepatocytes in the BM in vivo. This process may need some stimuli such as liver damage. In the present study, cell culture may have played a role in stimulation for differentiation of hepatic progenitor cells into hepatocytes. In addition, this process may depend on the HGF concentration, which is known to remarkably increase in the blood following liver damage (6). In any case, hepatic progenitor cells in the BM may be a useful resource for transplantation therapy for liver failure regardless of the major histocompatibility complex, since the patient’s own BM cells can be used for transplantation. Cultivation of BM cells in the presence of HGF has the following advantages: (i) it is possible to prepare a hepatocyte-enriched population from BM cells; (ii) it is possible to functionally improve BM-derived hepatocytes by introduction of important genes; and (iii) it is possible to develop a bioartificial liver device with BMderived hepatocytes. In addition, HGF is also useful for treatment of liver cirrhosis (29) and can induce differentiation of hepatic progenitor cells included in the BM into hepatocytes. Taken together, the combination of HGF and BM cells may be useful for treating liver diseases.
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