ToxSci Advance Access originally published online on July 13, 2006
Toxicological Sciences 2006 94(2):330-341; doi:10.1093/toxsci/kfl058
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Sequential Exposure to Cytokines Reflecting Embryogenesis: The Key for in vitro Differentiation of Adult Bone Marrow Stem Cells into Functional Hepatocyte-like Cells
* Department of Toxicology, Vrije Universiteit Brussel (VUB), B-1090 Brussels, Belgium
Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota 55455
Department of Analytical Chemistry, VUB, B-1090 Brussels, Belgium
1 To whom correspondence should be addressed at Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. Fax: +32 2 477 45 82. E-mail: sarah.snykers{at}vub.ac.be.
Received May 19, 2006; accepted July 5, 2006
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ABSTRACT |
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Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors (fibroblast growth factor-4, hepatocyte growth factor, insulin-transferrin-sodium selenite, and dexamethasone) in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis. Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked. Indeed, expression of the early hepatocyte markers alpha-fetoprotein and hepatocyte nuclear factor (HNF)3ß decreased as differentiation progressed, whereas levels of the late liver-specific markers albumin (ALB), cytokeratin (CK)18, and HNF1
were gradually upregulated. In contrast, cocktail treatment did not significantly alter the expression pattern of the hepatic markers. Moreover, sequentially exposed cells featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and inducible cytochrome P450–dependent activity, far more efficiently compared to the cocktail condition. In conclusion, sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult rat BMSC into functional hepatocyte-like cells. This model may not only be applicable for in vitro studies of endoderm differentiation but it also provides a "virtually unlimited" source of functional hepatocytes, suitable for preclinical pharmacological research and testing, and cell and organ development. Key Words: bone marrow stem cells; hepatocytes; sequential differentiation; liver-specific growth factors; liver embryonic development; in vitro.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Drug development is aimed at identifying pharmacologically active drug candidates with a favorable toxicologic profile. The increasing number of safety criteria, imposed on newly designed molecules, leads nowadays to the urgent need of in vitro techniques in the industry, developed according to the principle of Russell and Burch. To date, several hepatocyte-based in vitro models are available, however, they are not yet accepted into regulations, as they still require better characterization and optimization to reach the validation stage. Most primary hepatocyte cultures are in fact hampered by progressive occurrence of differentiation (De Smet et al., 2001
; LeCluyse et al., 1996; Rogiers and Vercruysse, 1993). An alternative approach would be the use of postnatal progenitor/stem cells.
Indeed, until recently, it was believed that tissue-specific stem cells could only differentiate into cells of the tissue of origin. However, a number of recent studies have suggested that adult stem cells may overcome germ lineage restrictions and express molecular characteristics of cells of different tissue origin, which has been termed "plasticity" (Jackson et al., 2001; Krause et al., 2001; Theise et al., 2000; Vourc'h et al., 2004). For example, hematopoietic cells may acquire characteristics of cardiomyocytes, cells of lung, gut, liver, blood vessels, skin, etc. (Jackson et al., 2001; Krause et al., 2001; Theise et al., 2000). This apparent plasticity can at least in some instances be explained by cell fusion (Wang et al., 2003). Other studies have described nonhematopoietic stem cells from bone marrow that are capable of differentiating in vitro in cells with mesodermal, ectodermal, and endodermal features (Jiang et al., 2002; Reyes et al., 2001; Yoon et al., 2005). The mechanism through which these cells gain multipotency is not totally understood (Verfaillie, 2000). Multipotent adult progenitor cells, for instance, can be induced to express phenotypic and functional characteristics of hepatocytes; however, the degree of differentiation obtained till now is incomplete (Schwartz et al., 2002).
Therefore, in order to develop an in vitro model suitable for pharmaco-toxicological purposes, attempts were made here to optimize the differentiation efficiency of nonhematopoietic stem cells from bone marrow into functional hepatocytes.
Liver development is accomplished by a sequential array of biological events. Each step of cell growth and differentiation is tightly regulated by cell autonomous mechanisms and extracellular signals, including cytokines and growth factors. More specifically, during the initial phase of murine liver ontogeny (embryonic days [E] 8–9), fibroblast growth factors (FGFs), derived from adjacent cardiac mesoderm, commend the foregut endoderm to form the liver primordium (Duncan, 2000; Jung et al., 1999). During and after the mid-stage of hepatogenesis, surrounding mesenchymal cells secrete hepatocyte growth factor (HGF) and support as such the fetal hepatocytes (Kinoshita and Miyajima, 2002; Zaret, 2002). Around E11, the fetal liver becomes the major site for hematopoiesis. During this stage, hematopoietic stem cells produce oncostatin M that, in the presence of glucocorticoids, not only promotes fetal hepatic cell differentiation and maturation but also suppresses embryonic hematopoiesis. In contrast, oncostatin M alone fails to induce differentiated liver phenotypes, implying that glucocorticoids are essential triggers for hepatic maturation (Kinoshita and Miyajima, 2002; Schmidt et al., 1995; Zaret, 2002). In rodents, the final step of hepatic differentiation takes place several days after birth. The lack of terminal differentiation of primary hepatocytes in culture evidences that additional signals, probably generated through the extracellular matrix, are necessary (Kinoshita and Miyajima, 2002).
Here, the liver development was taken as exemplar to establish a culture model that more readily supports robust differentiation of bone marrow stem cells (BMSC) to mature hepatocyte-like cells. We compared two experimental setups: (1) BMSC were treated with a cocktail of liver-specific factors (FGF-4, HGF, insulin-transferrin-sodium selenite [ITS], and dexamethasone [Dex]) as previously described (Schwartz et al., 2002) or (2) innovative in this field, BMSC were exposed to a sequence of these compounds in a manner that closely reflects their temporal expression during in vivo hepatogenesis (FGF-4, followed by HGF, followed by a combination of HGF, ITS, and Dex).
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MATERIALS AND METHODS |
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Isolation and culture of undifferentiated rat BMSC.
BMSC were isolated from male Fisher rats (4–6 weeks old) and cultured as described by Jiang et al. (2002)
. Labware used for expansion of BMSC included Corning 75 and 150 cm2 tissue culture flasks, polystyrene (both from VWR, Leuven, Belgium). Cell karyotyping, neuroectodermal, and endothelial differentiation were determined as previously described (Jiang et al., 2002, 2003; Reyes et al., 2001). Rats had access to food and water ad libitum and were housed according to guidelines from the Institutional Animal Care and Use Committee of the University of Minnesota.
Hepatocyte differentiation.
Rat BMSC from 60 population doublings on were used for differentiation into hepatocyte-like cells. BMSC were plated at 21 x 103 cells/cm2 on 1 mg/ml collagen type I–coated culture plates and dishes (BD Falcon 24-well plate, polystyrene; BD Falcon 35 x 10 mm petri dishes, polystyrene; NUNC F96 microwell plate, black, polystyrene; NUNC F96 microwell plate, clear, polystyrene [all from VWR]) in low-serum expansion medium (Jiang et al., 2002; Reyes et al., 2001). Once cells reached 100% confluence, they were washed with basal medium (Jiang et al., 2003) supplemented with 0.03mM nicotinamide, 0.25mM sodium-pyruvate and 1.623mM glutamine (all from Sigma, Bornem, Belgium). Subsequently, cells were cultured in the presence of liver-specific cytokines and growth factors, added either as a cocktail (basal medium + 10 ng/ml FGF-4, 20 ng/ml HGF [all from R&D Systems, Minneapolis, MN], 1 x ITS and 20 µg/l Dex [all from Sigma]) or sequentially (days 0–3: basal medium + 10 ng/ml FGF-4; days 3–6: basal medium + 20 ng/ml HGF; from day 6 on: basal medium + 20 ng/ml HGF + 1x ITS and 20 µg/l Dex). Differentiation media were changed every 3 days. A schematic presentation of the differentiation procedure is shown in Figure 1.
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Quantitative RT-PCR.
For PCR analysis, 1 µg RNA was reverse transcribed to cDNA using Superscript II reverse transcriptase and random hexamer primers (Invitrogen, Merelbeke, Belgium). The resulting RT-products were essentially amplified as previously described (Jiang et al., 2003
; Schwartz et al., 2002). Three extra steps were included to ensure the purity of the PCR products: 95°C for 15 s, 60°C for 20 s, and 95°C for 15 s. The primers used for amplification and the products expected are described in (Jiang et al., 2003; Schwartz et al., 2002). The RNA levels were normalized using 18S and compared with the RNA levels in undifferentiated BMSC (negative control) and freshly isolated primary rat hepatocytes (positive control). As a negative control for the primers, a no template cDNA-PCR reaction was run under the same conditions. The authenticity and size of the PCR products were confirmed by melting curve analysis (using software provided by Perkin Elmer, Lennik, Belgium) and gel electrophoresis.
Immunocytochemistry.
Differentiated BMSC were fixed either with ethanol for 10 min at – 20°C (cytoskeletal proteins) or with 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) for 10 min at 4°C, followed by incubation with 100mM glycin to saturate reactive groups (nuclear and cytoplasmic markers). The fixed cells were permeabilized for 15 min with 0.1% Triton in phosphate-buffered saline (Electron Microscopy Sciences) and blocked for 30 min with 1% bovine serum albumin/5% donkey serum block buffer at room temperature. After blocking, cells were incubated overnight at 4°C with primary antibody (fluorochrome-conjugated or nonconjugated) and washed three times with phosphate-buffered saline. In case the primary antibody was not conjugated, cells were incubated for 2 h at room temperature with secondary fluorochrome-conjugated antibody. After incubation, slides were washed again with 0.1% Triton in phosphate-buffered saline and mounted with Vectashield with DAPI (Vector Laboratories, Burlingame, CA). As a negative control, cells were incubated with appropriate gamma immunoglobulines (Jackson Immunoresearch, Cambridgeshire, UK) and immunostained under the same conditions. In order to evaluate the localization of cytochrome P450 (CYP) proteins, mitochondria and endoplasmic reticulum were counterstained with the carbocyanine dye DiOC6 (Molecular probes, Invitrogen). Cells were analyzed using fluorescence microscopy with a Zeiss Axiovert scope. To enumerate the number of cells expressing a given marker, all nuclei of positive-stained cells were counted and compared to the total number of cells evaluated. The primary antibodies against alpha-fetoprotein (AFP) (goat), hepatocyte nuclear factor (HNF)3ß (goat), and HNF1 (rabbit) were purchased from Santa Cruz, (Heidelberg, Germany). Anti-cytokeratin (CK)18 (mouse, FITC-conjugated) and anti-albumin (ALB) (goat, FITC-conjugated) antibodies were from Sigma and Bethyl Laboratories (Montgomery, TX), respectively. The antibodies against CYP1A1 and CYP2B1/2 (both goat) came from Daiichi pure chemicals, BD Biosciences (Tokyo, Japan). Respective secondary antibodies were purchased from Jackson Immunoresearch.
Albumin ELISA.
ALB concentrations, secreted into the culture media, were analyzed by ELISA (Koebe et al., 1994).
Urea assay.
The produced urea concentrations were, after 24-h exposure of the cells to 6mM NH4Cl, colometrically measured in culture media according to the manufacturer's instructions (Quantichrom Urea assay kit, Bioassay Systems, Brussels, Belgium). Fresh culture media supplemented with 6mM NH4Cl and 4 h-cultured adult rat hepatocytes were used as a negative and positive control, respectively.
Glycogen storage.
Intracellular glycogen was analyzed by Periodic-acid-Schiff staining (PAS-kit 395B-1KT, Sigma) according to the manufacturer's instructions. Amyloglucosidase (Sigma)-treated cells and 4 h-cultured adult rat hepatocytes were used as a negative and positive control, respectively.
Alkoxyresorufin-O-dealkylase assay.
Ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-dealkylase (PROD) activities were assessed as previously described (Donato et al., 1993) with some minor modifications: in our setup, cells were incubated with 20µM 7-ethoxyresorufin and 18µM 7-pentoxyresorufin (all from Sigma) for 30 min.
To evaluate the inducibility of CYP2B1/2 and CYP1A1/2, respectively, cells were, after 24 days of differentiation, exposed to phenobarbital (PB; final concentration 1mM) and 3-methylcholantrene (MC; final concentration 2µM; all from Sigma). Media, supplemented with either PB or MC, were daily renewed from that time on. Fresh culture media and 4 h-cultured adult rat hepatocytes were used as a negative and positive control, respectively.
Statistics.
Results are expressed as mean ± SD. Statistical analyses were performed using one-way ANOVA and Student's t-test. The significance level was set at 0.05.
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RESULTS |
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Characterization of the Differentiation Pattern of Rat BMSC into Hepatocyte-like Cells: Sequential versus Cocktail Exposure
Morphological Features
Previously, it has been shown that BMSC could differentiate into hepatocyte-like cells upon simultaneous exposure to a mixture of well-defined cytokines and growth factors (Schwartz et al., 2002
). However, using this approach, a rather heterogeneous population of epithelioid cells and other cell types was obtained. Moreover, no polygonal-shaped cells and only few binucleated cells were formed (Fig. 2). In an attempt to improve the differentiation of nonhaematopoietic stem cells from bone marrow into hepatocyte-like cells, BMSC were exposed to the same well-defined hepatogenic factors, but in a sequential way. More specifically, cytokines and growth factors were added at defined points in time, in a manner that closely resembles the in vivo process of embryonic liver development as specified in "Materials and Methods" section (Duncan, 2000; Jung et al., 1999; Kinoshita and Miyajima, 2002; Schmidt et al., 1995; Zaret, 2002). In this novel setup, epithelioid cells appeared in culture from day 6 on (Fig. 2). However, at that moment these cells were still surrounded by spindle-shaped cells. After 14 days, less fibroblastic cells were seen and some binucleated cells appeared. After 18 days, most cells exhibited a polygonal shape (Fig. 2).
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Characterization at the Molecular Level
In a next set of experiments, we evaluated whether these morphological differences were associated with distinct patterns of differentiation at the molecular level. Therefore, the expressions of early (AFP and HNF3ß) and late (ALB, CK18, and HNF1
) liver-specific markers were analyzed at both the mRNA (Fig. 3) and protein levels (Figs. 4 and 5).
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mRNA expression.
In both sequential and cocktail culture conditions, AFP, HNF3ß, ALB, CK18, and HNF1
were expressed in a time-dependent manner during BMSC differentiation. Both the pattern and the level of expression, however, differed considerably between the culture methods. In fact, upon sequential exposure to liver-specific factors, maximal AFP mRNA expression occurred after 6 days (Fig. 3), 4 days later than seen in the cocktail condition, but was 1.2-fold higher than the maximal level observed in cocktail-exposed cells. AFP mRNA expression disappeared completely in both conditions by day 11 of culture. In sequentially treated cells, downregulation of AFP mRNA expression was nicely followed by a second transient, though more pronounced, induction of the early liver-specific marker HNF3ß as well as by a steady upregulation of the late hepatic markers ALB, CK18, and HNF1 (Fig. 3). More specifically, HNF3ß mRNA expression started at day 2, reached maximal levels at day 10 and decreased rapidly thereafter (Fig. 3). ALB and CK18 mRNA expression, on the other hand, gradually increased from days 4 and 10 of culture, respectively, until maximal levels were reached at day 18 (Fig. 3). In sharp contrast to these observations, changes in HNF3ß, ALB, and CK18 mRNA levels were negligible upon simultaneous exposure to all hepatogenic factors (p < 0.001; one-way ANOVA). In addition, the mRNA of the late liver-specific marker ALB remained very low in cocktail-exposed BMSC, suggesting an immature hepatic differentiation status. Finally, upon sequential exposure, HNF1 mRNA expression gradually increased from day 6 on whereas in the cocktail condition, HNF1 mRNA induction was delayed by 4 days and occurred only transiently (Fig. 3). Moreover, maximal levels, obtained at day 12, were about twofold lower than the levels observed in 12-day–old BMSC in the sequential condition. Thus, sequentially exposed BMSC underwent a consecutive array of developmental stages comparable with in vivo hepatogenesis while exposure to a cocktail of cytokines and growth factors induced an aberrant expression pattern of differentiation when compared to liver embryogenesis.
Protein expression.
In order to support the results obtained at the mRNA level, immunocytochemistry analyses were performed in parallel (Figs. 4 and 5). After 4 days of differentiation, cells expressed AFP, regardless of the experimental setup (Figs. 4 and 5). Concomitantly to the results found at the mRNA level (Fig. 3), AFP expression occurred only transiently in both conditions (Figs. 4 and 5) and was undetectable by day 12 of culture (data not shown). Upon sequential exposure to liver-specific factors, a maximal positive staining of HNF3ß (92 ± 8%) was noticed at day 10, leveling off thereafter (Fig. 4). Treatment with all factors simultaneously, however, revealed no more than 24 ± 7% HNF3ß-positive cells throughout the culture period (Fig. 5). As differentiation progressed, extensively increased stainings for ALB, CK18, and HNF1 were detected upon sequential exposure to cytokines and growth factors, in accordance with the results obtained at the RNA level (Figs. 3 and 4). Consequently, after 18 days, 92 ± 2%, 94 ± 3%, and 89 ± 9% of the cells, respectively, stained positive for these markers (Fig. 4), which is in sharp contrast to only 32 ± 4%, 63 ± 5%, and 22 ± 4% of the cocktail-exposed cells, respectively (p < 0.001; Student's t-test) (Fig. 4).
In addition, in order to state the immunocytochemistry data with certainty, immunoblotting has been performed in parallel once (data not shown). In line with the previous results obtained at both the mRNA and protein level, sequentially exposed cells expressed liver-specific proteins more abundantly than cells in the cocktail setup. However, since this approach consumes large numbers of cells, i.e., at least 25–50 µg of protein is needed to analyze one liver-specific marker at one point in time, the analysis was not repeated. Alternatively, as measuring CYP activity (the set of EROD/PROD) and their inducibility are widely accepted as final end point to evaluate the suitability of cells as in vitro models for pharmaco-toxicological screening of drugs (De Smet et al., 2001; Donato et al., 1993, 2003; LeCluyse et al., 1996; Rogiers and Vercruysse, 1993), we enlarged, in a next set of experiments, the data set on cell functionality in order to increase confidence in our data.
Hepatic Functionality
In order to assess whether these hepatocyte-like cells derived from the bone marrow also acquired typical functional hepatic features, ALB secretion, ammonia metabolism, glycogen storage, expression of CYP proteins in parallel with their activity and inducibility were evaluated.
ALB secretion.
Sequentially treated BMSC significantly upregulated the ALB secretion rate from day 15 onward (p < 0.01, Student's t-test) (Fig. 6). On the contrary, BMSC exposed to a cocktail of liver-specific factors did not secrete ALB above basal levels, corresponding to 0.55 µg/ml (Fig. 6).
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Ureogenesis.
Upon sequential exposure to hepatogenic factors, the urea production increased over culture time, reaching adult levels after 30–33 days. In contrast, cocktail-exposed cells synthesized, even at peak production, 24% significant lower urea levels (p < 0.05; Student's t-test) (Fig. 7).
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Glycogen storage.
Furthermore, upon sequential treatment with cytokines and growth factors, glycogen uptake was first seen after 21 days of culture, 6 days earlier than in the cocktail condition. After 30 days of culture, about 86% of the cells stored glycogen, regardless of the culture method (Fig. 8).
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CYP protein-expression, activity, and inducibility.
In the sequential setup, phase I CYP1A1 and CYP2B1/2 proteins were expressed within and nearby the endoplasmic reticulum and mitochondria (Fig. 9). The level of expression gradually increased as differentiation progressed. After 30 days, 78 ± 1 and 79 ± 3% of the cells stained positive for CYP1A1 and CYP2B1/2, respectively (Fig. 10). In sharp contrast to these observations, cocktail-exposed cells only showed modest CYP expression over culture time (Figs. 9 and 10).
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In addition, we investigated whether CYP1A1 and 2B1/2 were functionally active by measuring the respective EROD and PROD activities in both conditions (Figs. 11 and 12).
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In line with the results found at the protein level, sequentially exposed cells exhibited markedly higher EROD and PROD activity rates compared to the cocktail model (p < 0.05 at days 36 and 39; Student's t-test) (Figs. 11 and 12). Upon sequential exposure to liver-specific factors, a transient fourfold increase in PROD activity was displayed by days 27– 30, approaching the level of 4 h-cultured adult rat hepatocytes, versus only a twofold increase after cocktail treatment (Fig. 11). In addition, in the former setup, EROD activity gradually increased from days 27 to 36 towards levels measured in 4 h-cultured adult rat hepatocytes, whereas CYP1A1/2-dependent activities appeared only transiently in cocktail-exposed cells between days 30 and 33 and declined to almost nondetectable levels on day 36 (Fig. 12).
CYP-inducibility is considered as the most representative metabolic function of the adult hepatic phenotype (Gomez-Lechon et al., 2004; Rogiers and Vercruysse, 1993). Therefore, the responsiveness of both CYP1A1/2 and CYP2B1/2 to their respective prototype inducers MC and PB was analyzed in parallel. PROD activities were induced up to 1.4-fold after 6-day exposure to PB (i.e., on day 30), regardless of the experimental setup (Fig. 11). The inducibility persisted for 6 days in sequentially exposed cells but not in the cocktail condition. A significant CYP1A1/2-dependent response to MC was observed on days 36–39 (p < 0.001 and p < 0.01 at days 36 and 39, respectively; Student's t-test) in the sequential model. Conversely, MC barely induced EROD activities upon culture with all liver-specific factors simultaneously (Fig. 12).
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DISCUSSION |
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In recent years, adult-derived stem cells have become a hot topic in the field of molecular, cellular, and clinical biology, as well as in pharmaco-toxicology. Indeed, stem cells have an extensive self-renewing potential and many of them are considered multipotent (Jackson et al., 2001
; Krause et al., 2001; Theise et al., 2000; Vourc'h et al., 2004). This interest in adult stem cells has in particular been triggered by the numerous ethical dilemmas surrounding the use of embryonic stem cells in preclinical and clinical research (Henningson et al., 2003; McLaren, 2001). The best-characterized stem cell compartment is the bone marrow consisting of two stem cell populations, referred to as the hematopoietic and the mesenchymal stem cells (Huttmann et al., 2003). Previously, Schwartz et al. (2002) described a population of cells in postnatal rat bone marrow, copurified with mesenchymal stem cells, that were capable of differentiating into cells of endodermal (hepatocytes) origin upon exposure to well-defined hepatogenic factors. These culture conditions yielded, however, a mixture of epithelioid cells and other cell types. Therefore, attempts were made here to improve the hepatic differentiation process through exposure of BMSC to the same liver-specific factors in a sequential time-dependent manner, reflecting their secretion during in vivo hepatogenesis (Duncan, 2000; Jung et al., 1999; Kinoshita and Miyajima, 2002; Schmidt et al., 1995; Zaret, 2002).
Under these culture conditions, BMSC acquired morphological features (polygonal-shaped and binucleated cells) similar to those of primary hepatocytes (Ferrini et al., 1997; Katsura et al., 2002). Furthermore, more than 85% of these epithelioid cells expressed liver-associated genes and proteins (AFP, HNF3ß, ALB, CK18, and HNF1) in a comparable time-dependent manner as observed during in vivo liver embryogenesis. Indeed, AFP expression is first detected in embryonic endoderm around E8.5 (Cascio and Zaret, 1991) and precedes ALB and HNF1 expression, detected around E9.5 and E10.5, respectively (Ott et al., 1991; Shiojiri, 1981). This finding implicates that, in this setup, the BMSC differentiation process could serve as a model of early mammalian endoderm differentiation. In contrast, upon exposure to a cytokine/growth factors-cocktail, the expression patterns differed from the normal sequence seen during in vivo hepatogenesis as HNF1 expression preceded that of ALB. Indeed, HNF1 is only expressed in fully differentiated cells and not in un- or dedifferentiated cells (Cereghini et al., 1988), as was noticed here upon cocktail treatment. In addition, significantly lower levels of liver-specific markers were expressed. The higher levels of ALB and CK18 expression in the sequential condition are probably due to the higher levels of both the early (HNF3ß) and late (HNF1) transcription factors. It is well documented that liver-enriched transcription factors act cooperatively and synergistically to promote liver-specific gene transcription (Cereghini et al., 1992; Darlington, 1999; Duncan, 2000; Hayashi et al., 1999; Shim et al., 1988). In this regard, it was previously shown that HNF3ß positively regulates the expression of HNF4 and HNF1 (Darlington, 1999; Duncan et al., 1998). Furthermore, it is believed that HNF3ß serves as the initiator of a cascade of regulatory events resulting in endoderm induction (Ang et al., 1993; Darlington, 1999; Duncan, 2000; Levinson-Dushnik and Benvenisty, 1997). Hence, the minor changes in HNF3ß expression levels in the cocktail condition may only result in low levels of ALB and CK18 transcripts and protein.
The initiation and induction of AFP expression is not yet completely understood. It can be assumed that additional factors are involved in its transcriptional activation, as in both culture conditions, only minimal levels of HNF3ß were detected at the time of AFP expression. Further research will be needed to fully elucidate the transcriptional hierarchy mediating differentiation of BMSC toward hepatocytes.
The presence of both morphologic and phenotypic features, similar to that of primary hepatocytes, does, however, not fully prove the differentiation of BMSC into mature hepatocytes. Indeed, during the terminal step of liver organogenesis, the liver becomes a functional and metabolic organ, performing an essential role as detoxifying center of the body (Gomez-Lechon et al., 2004; Kinoshita and Miyajima, 2002; Zaret, 2002). Interestingly, functional maturation occurred in both experimental setups, but to a different extent. Hepatic metabolic functions, including ALB secretion, urea production, storage of glycogen, and CYP-activity/inducibility, were manifested most prominently upon sequential exposure to hepatogenic factors. Under these culture conditions, ALB secretion was in fact significantly upregulated to levels comparable to those obtained in both 2- to 7-day–old immobilization and 7-day–old monolayer cultures of primary rat hepatocytes. The latter measurements are performed on a regular basis in our laboratory (Beken et al., 2001; Vanhaecke et al., 2004). In addition, both the urea production and EROD/PROD activities reached levels comparable to 4 h-cultured primary rat hepatocytes. Response to prototype inducers was as expected: pronounced upon exposure to MC and discrete upon PB treatment. The level of induction, however, remained lower in comparison to cultured adult rat hepatocytes. More specifically, EROD activity increased up to fourfold after 15-day exposure to MC in sequentially exposed BMSC versus maximal sevenfold in 2-day treated rat hepatocytes (Donato et al., 1993). Nevertheless, to our best knowledge, this is the first time that EROD (CYP1A1/2) activity/inducibility is demonstrated in hepatocyte-like cells derived from BMSC.
The less mature phenotype of cocktail-exposed cells could possibly be ascribed to altered and lower expression of HNF-type liver-enriched transcription factors in this setup. Experiments using hepatoma cell lines and HNF-null mice have in fact demonstrated the important role of HNFs in the regulation of genes that are involved in biotransformation (Cyps) and ammonia metabolism (ornithine-transcarbamylase gene) (Gomez-Lechon et al., 2004; Inoue et al., 2002; Rodriguez-Antona et al., 2002). Similar to the results reported here, inducible CYP2B1/2-activity was also found by Schwartz et al. (2002) after exposure to a cocktail of the same cytokines and growth factors, although at an earlier time in culture. Some variation in time-specific gene and protein expression could probably be attributated to intraspecies differences and subtle changes in the differentiation procedure (i.e., type of culture plate coating, serum, etc.).
In summary, during the first 18 days of the hepatic differentiation process of BMSC, cells, and sequentially exposed BMSC in specific, underwent a sequential array of developmental stages, characterized by the down- and upregulation of early and late liver-specific markers, respectively. As differentiation progressed, i.e., from day 18 onward, expression of mature hepatic markers persisted at steady levels (data not shown) and cells gradually underwent functional hepatic maturation. In specific, sequentially treated BMSC accomplished hepatic functions at levels comparable to those of primary rat hepatocytes, cultured for 4 h to 2 days. Our results thus clearly show that a more pronounced and homogeneous differentiation of BMSC into functional hepatocyte-like cells can be obtained by sequentially directing the differentiation process analogous to liver embryogenesis. Moreover, differentiation appears to occur via steps commonly defined for in vivo endodermal lineage specification and subsequent hepatocyte differentiation and maturation. Further investigations, in order to elucidate the molecular mechanisms underlying the changes described herein, are underway.
This model opens new perspectives: it may not only be applicable to study endoderm differentiation in vitro but it also offers the possibility to purify and culture multipotent stem cells from nonembryonic origin as an unlimited cell source for pharmaco-toxicological research and testing, and cell and organ development. It might even open a road to trigger cell fate and "trans"-differentiate uncommitted cells from different tissues towards endodermal lineages.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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The authors thank B. Dejaegher for the valuable statistical advice and L. Grooten, B. Degreef, E. Desmedt, S. Coppens, W. Sonck, Y. Heremans, and M. Blackstad for their dedicated technical assistance. Research was funded by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (Instituut voor de aanmoediging van Innovatie door wetenschap en technologie in Vlaanderen) and grants from the Research Council (Onderzoeksraad) of the Vrije Universiteit Brussel, Belgium, and Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek in Vlaanderen), Belgium. The project is also a part of the EU FP6 project, Lintop.
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