Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow
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The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated.
Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity.
hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.
KeywordsHepatocyte Growth Factor HDAC Inhibition Bone Marrow Sample Becton Dickinson Hepatic Differentiation
CCAAT-enhancer binding protein
fibroblast growth factor-4
hepatocyte growth factor
hepatocyte nuclear factor
histone deacetylase inhibitor
human adipose tissue-derived stromal cells
human mesenchymal stem cells
multidrug resistance-associated protein
multipotent adult progenitor cells
new chemical entities
The ever-increasing number of new chemical entities (NCEs) has prompted the development of research methods for rapid screening. Present research efforts are therefore directed at developing a series of in vitro assays that could be applied for preclinical screening of NCEs. By utilizing in vitro models, pharmaceutical companies attempt to reduce clinical failure rates by accurately evaluating efficacy and safety much earlier in the drug discovery process . As the liver is the principal organ for xenobiotic biotransformation and thus a key target for drug-induced toxicity, in vitro models to predict drug metabolism are preferably derived from human liver tissues. However, the use of human hepatocytes is limited by scarcity of intact and fresh human liver tissue [2, 3]. In addition, primary hepatocyte cultures still cope with a progressive occurrence of dedifferentiation, resulting in a relative short life-span and a rapid decline of liver-specific functions [4, 5, 6, 7]. Different alternatives have been explored to overcome these difficulties, including the use of human stem cells. In vivo as well as in vitro studies have provided evidence that stem cells can overcome germ lineage restrictions and express molecular characteristics of cells of different tissue origin [8, 9].
The best characterized stem cell compartment is the bone marrow consisting of two stem cell populations, referred to as the hematopoietic and mesenchymal stem cells . The latter was first described by Friedenstein et al. as a population of cells isolated from the bone marrow and capable of differentiation into bone, adipocytes, chondrocytes, osteoblasts, osteoprogenitors, skeletal myocytes, tendon and bone marrow stromal cells [10, 11].
Schwartz et al. described a novel population of cells within postnatal rat bone marrow, named multipotent adult progenitor cells (MAPC), that were not only capable to differentiate into most mesodermal cell types, but also into functional neuroectodermal and endodermal (hepatocytes) cell types .
In the present study, we investigated whether hMSC also have the potential to differentiate in vitro into functional hepatocytes. Therefore, hMSC were cultivated in the presence of 'hepatogenic' factors, added either as a cocktail (FGF-4, HGF, ITS and dexamethasone) or, more innovative, in a sequential manner closely reflecting their temporal expression during in vivo hepatogenesis (FGF-4, followed by HGF, followed by a combination of HGF, ITS and dexamethasone). Next, giving the promising differentiation-inducing properties of histone deaceylase inhibitors (HDAC-I) in primary cultures of adult hepatocytes [13, 14, 15, 16] and hepatoma cell lines [17, 18, 19], we additionally examined the effect of TSA, a potent and specific hydroxamic acid-based HDAC-I, on hMSC cultures.
Characterization of culture-expanded hMSC
hMSC were cultivated from the mononuclear cell fraction of bone marrow samples obtained from healthy donors. To ensure removal of contaminating hematopoietic cells, cells were selected, based on plastic adherence, and passaged 4 times prior to further use.
Differentiation of hMSC under 'hepatogenic-supporting' conditions
To evaluate whether hMSC could differentiate into endodermal cells, as described earlier for MAPC , cells were exposed to hepatogenic agents, either as a cocktail  or in a sequential manner , and analyzed by means of microscopic analysis and specific cytological stainings.
Optimization of culture conditions
A prerequisite for differentiation in vitro is cell cycle arrest. In preliminary experiments, we found that, in the presence of cytokines and growth factors, a non-proliferative status is only obtained at 100% confluence. Therefore, the latter cell density was further applied in all differentiation experiments.
Endogenic differentiation upon sequential exposure of hMSC to hepatogenic factors
TSA, a trigger for functional maturation of endogenic differentiating hMSC
In an attempt to enhance endogenic differentiation of hMSC, TSA, a selective and reversible HDAC-I [13, 14, 15, 16, 17, 18, 19], was added to the culture media in a concentration of 1 μM after exposure of the cells to the hepatogenic factors.
Optimization of culture conditions
Besides the state of growth arrest, 'time' is often critical for successful 'trans'differentiation of (progenitor/stem) cells in epigenetic regulating processes . More specifically, promotion of 'trans'differentiation into a specific lineage by means of HDAC inhibition often requires selective pre-stimulation of the cells. In preliminary experiments, we searched for the most optimal differentiation conditions. Hereto, we examined the effect of the hepatic inducing effects of (1) FGF-4, HGF, ITS and dexamethasone, added alone and in different combinations (e.g. FGF-4, HGF/HGF, ITS/HGF, dexamethasone/ITS, dexamethasone/ITS, HGF etc.), and (2) pre- and post-treated with the HDAC inhibitor TSA. Out of the results obtained, we found that 1 μM TSA, when added exclusively to MSC at 100% confluence and without pretreatment with FGF-4 and HGF, was not effective in stimulating mesenchymal-to-hepatic transition. The same applies when these cells were treated with FGF-4 and HGF afterwards (data not shown). In addition, treatment of undifferentiated hMSC with 1 μM TSA resulted in massive cell death and cell detachment (data not shown). Suppression of proliferation, in parallel with cell survival and long-term cultivation, were only obtained upon pre-stimulation of the cells with hepatogenic factors for at least 6 days prior to addition of 1 μM TSA (see Additional files 1, 2, 3). In spite of the good long-term cell survival, about 20% of the TSA-treated cells underwent apoptosis (see Additional file 3). In the present study, the most appropriate conditions for transdifferentiation comprised exposure of hMSC to 1 μM TSA, starting on the 6 th day of exposure to hepatogenic cytokines and growth factors; these treatments are further referred to as 'cocktail TSA' and 'sequential TSA' conditions.
Liver-specific protein expression
To evaluate whether these hepatocyte-like cells derived from adult human bone marrow also acquired typical functional hepatic features, ALB secretion, ammonia metabolism and inducible CYP-dependent activity were next analysed.
CYP-dependent activity and inducibility
The potential to induce CYP-dependent monooxygenases is considered as one of the most representative functional parameters to evaluate the adult hepatic phenotype [22, 23]. Therefore, the responsiveness of both CYP1A1/2 and CYP2B6 to their respective prototype inducers 3-methylcholantrene (MC) and phenobarbital (PB) was studied in parallel. Interestingly, only TSA-treated cells exhibited inducible CYP-activities (Figs. 8, 9). More specifically, upon TSA-treatment pentoxyresorufin-O-dealkylase (PROD)-activities were significantly induced up to 2.2 fold after 3 days exposure to PB (i.e. on day 21), regardless of the experimental configuration (p < 0.05; Student's t-test). The inducibility persisted throughout the culture time (Fig. 8). In addition, a significant 6.4 fold CYP1A1/2-dependent response to MC was observed on days 24–27 in the sequential TSA-model (p < 0.05; Student's t-test). Cocktail TSA-treated cells also showed inducible EROD-activity, but to a significantly lesser extent (p < 0.001, Student's t test) (Fig. 9).
Summary of all characterization experiments performed along with their outcome.
AFP, HNF3β, ALB, CK18, HNF1α, C/EBPα, MRP2
Inducible EROD activity
Inducible PROD activity
TSA causes accumulation of acetylated histone H4
The question, however, still remains whether the improved hepatic differentiation of hMSC, upon exposure to 1 μM TSA, is mediated by TSA-induced HDAC inhibition. Since histone H4 is a known target of TSA (21), the acetylated status of this protein was analysed by means of immunofluorescence.
Hepatocyte-based in vitro models are often-used tools for pharmaco-toxicological research and could also become an interesting tool for regulatory testing of xenobiotics . A serious drawback, however, is the occurrence of cellular dedifferentiation [5, 6, 7] and the shortage of cells of human origin. The use of stem cells could offer a solution. It is well recognized that in adult mammals, including humans, a number of tissues are continuously regenerated from immature cells (i.e. skin, intestinal epithelia, blood, the olfactory bulb in the brain) [8, 9, 10, 11, 12, 24, 25]. The 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 . Consequently, successful isolation, cultivation and manipulation of multipotent stem cells from non-embryonic origin could provide researchers with an unlimited human cell source for cell and organ development studies, preclinical pharmaco-toxicological research and regulatory testing.
In the current study, we investigated the in vitro endodermal potency of hMSC. hMSC were hereto exposed to well-defined hepatogenic agents, added either as a cocktail ('cocktail condition') or, more innovative, in a sequential time-dependent order as seen during liver embryogenesis ('sequential condition').
Exposure of hMSC to a cocktail of hepatogenic factors failed to induce mesenchymal-to-epithelial transition. In contrast, sequential treatment with these hepatogenic agents ('sequential condition') not only induced expression of the mid-late hepatic marker CK18 from day 8 onwards but also resulted in hepatic functionality, as evidenced by upregulated glycogen storage from days 8 to 22 and acquisition of CYP2B6-activity, though not inducible, from day 21 onwards. However, cells did not morphologically resemble adult hepatocytes.
As a trigger for further differentiation of hMSC towards the endodermal lineage, TSA, known as a proliferation-inhibiting and differentiation-inducing agent in primary hepatocytes and hepatoma cell lines [14, 15, 16, 17, 18, 19], was introduced into the present culture media. Previously, our group showed that exposure of 1 μM TSA to primary cultured hepatocytes induced cell cycle arrest during G0/G1 and G1/S phase in EGF-stimulated cells, significantly retarded apoptosis and improved cell junction intercellular communication and xenobiotic biotransformation [13, 14, 15, 16, 27, 28]. These findings encouraged us to expose differentiating hMSC to TSA as well. In fact, upon addition of TSA, hMSC of 1/4 of the bone marrow samples tested, underwent a mesenchymal-to-hepatic transition. More specifically, differentiating TSA-exposed cells adopted an epithelial morphology, expressed liver-specific proteins and acquired functional maturation, regardless of the experimental set-up, although to a different degree. A rather homogeneous population of epithelioid cells was obtained upon sequential TSA-treatment. About 70% of these differentiated cells expressed early (HNF3β, AFP), mid-late (ALB, CK18) and late (HNF1α, MRP2 and C/EBPα) hepatocyte markers in a ranking order comparable to their expression pattern during in vivo hepatogenesis. Indeed, AFP expression is first detected in embryonic endoderm around E8.5  and precedes ALB, HNF1α and MRP2 expression, detected around E9.5, E10.5 and E16, respectively [30, 31, 32].
Furthermore, hepatic metabolic functions, including ALB secretion, urea production, and inducible CYP-activity, were manifested exclusively in TSA-treated cells and most prominently under the sequential TSA-conditions. More specifically, TSA significantly upregulated ALB secretion, urea synthesis and CYP1A1/2-activities to levels comparable to those found in 4 hours- to 2 days-old monolayer cultures of adult hepatocytes . In line with the behaviour of adult hepatocytes, both in vitro and in vivo, responses to the prototype inducers MC and PB were pronounced and discrete, respectively; however, levels of induction, measured in the bone marrow-derived hepatocyte-like cells remained lower than in true adult hepatocytes.
On the other hand, cocktail TSA-treated cells also featured hepatic phenotype and function, but less differentiated, which corresponds well to the persistence of processes throughout the culture time.
Recently, the hepatic differentiation potential of MSC, derived from bone marrow, adipose tissue and umbilical cord blood, has been affirmed by showing expression of distinct hepatocyte markers and functions, including ALB and urea secretion, glycogen storage and LDL uptake, upon in vitro conditioning (i.e. exposure to hepatogenic agents) of the cells [33, 34, 35, 36, 37, 38, 39, 40, 41]. In contrast to our findings, a functional hepatic phenotype was generally not seen before 3.5–4 weeks post-stimulation. In addition, no inducible CYP-dependent activity was reported, which is considered to be a key determinant of the functional hepatic phenotype, and a prerequisite for applicability of the cells as in vitro models for preclinical drug testing. Only one study described inducible Cyp2B6 expression at both the mRNA and protein-level  but not before 4 to 6 weeks preconditioning of the cells . In contrast, our TSA-exposed cells already possessed PROD (CYP2B6)-dependent activity and -inducibility upon 21 days differentiation.
In line with our results, Seo et al., 2005 showed enhanced hepatic differentiation upon addition of 0.1% dimethylsulfoxide (DMSO) to human adipose tissue-derived stromal cells (hADSC), prestimulated for 10 days with a cocktail of hepatogenic cytokines . The mechanism underlying the hepatic (trans)differentiation-inducing effects of DMSO have not yet been fully elucidated. However, like TSA, histone hyperacetylation-inducing effects have been ascribed to DMSO as well .
Other research groups showed hepatic differentation of hMSC upon stimulation to hepatogenic cytokines exclusively, added either as a mixture [FGF-4+HGF [35, 36], separately [HGF ; HGF followed by oncostatin M (OSM) [34, 37] or as a combination thereof [FGF+HGF followed by OSM ]. Conform to our experience, the results shown by Lange et al., 2005 disagreed with those reports . The reasons underlying these controversial results are unknown. However, part might be ascribed to subtle differences in hMSC isolation/expansion techniques, and microenvironment (growth factors, cytokines, medium additives, cell-cell/cell-matrix contact, etc.), playing a decisive role in the specific and directed differentiation of stem/progenitor cells. Additionally, it should be taken into consideration that the plasticity of stem/progenitor cells highly depends on the tissue harvest site and the donor's profile (age, gender, body mass index, lifestyle, drug intake, etc.). The differentiating potential of bone marrow-derived MSC, in fact, decreases with age . In our study, most bone marrow samples were harvested from elderly patients, which might also form a part of the answer. In addition, the hepatic differentiation efficiency of all bone marrow samples tested in the present study yielded 25%, postulating influence of donor-related variability as well. The elucidation of all factors involved seems thus of primary importance and will be explored in future experiments.
The developmental process of hepatocytes towards a fully polarized and functional phenotype is known to depend on a complex network of transcriptional and posttranscriptional regulatory events. To our best knowledge, the results presented in this paper (patent submission PCT-EP2004-0012134) are the first to indicate that HDAC inhibition is, next to acquiring and maintaining differentiated functions in primary cells and cell lines, a key-factor to differentiate bone marrow-derived hMSC into functional hepatocyte-like cells, particularly under sequential culture conditions mimicking in vivo hepatogenesis.
Long-term cultivation and successful transdifferentiation of hMSC could be shown under both set-ups (sequential TSA and cocktail TSA), though a relatively high number (about 20%) of TSA-treated cells died, due to apoptosis. A feasible explanation could be that TSA, accompanied by hepatogenic agents, promotes selective survival of differentiating cells towards the hepatocyte lineage by inducing apoptosis of non- (hepatic) differentiating cells.
The established model opens new perspectives: it offers an unlimited source of functional human hepatocyte-like cells of non-embryonic origin, applicable in basic research (developmental endoderm biology), preclinical drug discovery and development, and cell and organ design. It might open a road for 'trans'-differentiation of cell types from different tissues towards endodermal lineages. In addition, as a consequence of their extensive self-renewing capacity, these cells may, in future, overcome the shortage of human donor tissue contributing as such to tissue regeneration in congenital and degenerative disorders.
Expansion of hMSC
Bone marrow samples were aspirated from the sternum of healthy (male and female) donors with variable age (35–85 years old), following informed consent. Bone marrow mononucleated cells were isolated by Ficoll-Hypaque (Nycomed, Lucron Bioproducts, De Pinte, Belgium) density-gradient centrifugation and plated in Mesenchymal Stem Cell Growth Medium (MSCGM, Cambrex, Verviers, Belgium) at 37°C and under 5%CO2 and 95%air After 4 hours, medium was renewed to remove non-adherent cells. At 50% confluency, cells were detached with trypsin (0.05% w/v)/EDTA (0.02% w/v) (Cambrex). Cells were cultured for 4 passages and finally harvested for further experiments.
Characterization of culture-expanded hMSC
Cells were labeled with CD34-FITC [Becton Dickinson (BD), Erembodegem, Belgium], CD45-PE (DAKO), CD105-FITC (Ancell-10P's, Zandhoven, Belgium), CD73-PE, CD90-PE, CD166-PE (all from PharMingen, BD) and analyzed using a flow cytometer (Coulter Epics® XL-MCL, Analis, Namur, Belgium).
Routine analysis of mesodermal potency
Cells were cultivated in specific media for adipogenic, osteogenic and chrondrogenic differentiation according to the manufacturer's instructions (Cambrex). Mesodermal differentiation was verified by means of different stainings (described below). To exclude spontaneous differentiation, hMSC were cultivated in regular MSCGM medium (Cambrex) and analyzed in parallel.
Endodermal (hepatocyte) differentiation of hMSC
hMSC were plated at 21.5 103 cells/cm2 on 1 mg/ml collagen gel type I (BD) in basal medium supplemented with 2% (v/v) foetal bovine serum (Hyclone, Perbio Science, Erembodegem, Belgium). Basal medium consisted of 60% (v/v) DMEM and 40% (v/v) MCDB-201 (Invitrogen, Merelbeke, Belgium) supplemented with 100 IE/ml penicillin (Continental Pharma, Diegem, Belgium), 100 μg/ml streptomycin, 1 mg/ml linoleic-acid bovine serum albumin, 0.1 mM L-ascorbic acid, 0.03 mM nicotinamide, 0.25 mM sodium pyruvate and 1.623 mM glutamine (all from Sigma, Bornem, Belgium). At 100% confluence, cells were exposed to hepatogenic cytokines and growth factors, added either simultaneous as a cocktail ('referred to as 'cocktail-condition/-set-up/-exposure' or a derivative thereof) [basal medium + 10 ng/ml FGF-4, 20 ng/ml HGF (all from R&D Systems, Minneapolis, USA), 1 × ITS and 20 μg/l dexamethasone (all from Sigma)] or sequentially ('referred to as 'sequential-condition/-set-up/-exposure' or a derivative thereof) (days 0–2: basal medium + 10 ng/ml FGF-4; days 3–5: basal medium + 20 ng/ml HGF; from day 6 on: basal medium + 20 ng/ml HGF + 1 × ITS and 20 μg/l dexamethasone). Differentiation media were changed every 3 days. From day 6 onwards, 1 μM TSA (Sigma) was added. The cocktail- and sequential-conditions, supplemented with 1 μM TSA will be further referred to as 'cocktail TSA-condition/-set-up/-exposure', and 'sequential TSA condition/-set-up/-exposure, respectively, or a derivative hereof.
Cells were fixed with 10% (w/v) formalin (PolySciences, Omnilabo International N.V. Aartselaar, Belgium) for 10 min at room temperature (adipogenic, hepatocyte differentiation) or with methanol (Merck, Overijse, Belgium) for 2 min at -20°C (osteogenic differentiation). After fixation, adipocytes were identified as red-colored lipid droplets upon staining with Oil-red O (from Sigma) . Mineralized nodules or calcium deposits in differentiated osteogenic cells were stained black with the von Kossa technique . Periodic-acid-Schiff (PAS) staining was used to determine glycogen storage, a functional parameter of endogenic differentiation . As a control, PAS staining was performed in the presence of amyloglucosidase (Sigma).
Chondrocytic differentiation was determined using rabbit polyclonal anti-collagen II antibody (NCL-COLL-IIp, NovoCastra, Prosan, Merelbeke, Belgium).
Cells were fixed either with ethanol (Merck) for 10 min at -20°C (cytoskeletal proteins) or with 4% (w/v) paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) for 10 min at 4°C (nuclear and cytoplasmic markers). After fixation, liver-specific protein expression was analysed using primary antibodies against AFP (goat), (HNF3β (goat), HNF1α (rabbit), C/EBPα (rabbit), MRP2 (goat; all from Santa Cruz, Heidelberg, Germany), CK18 (mouse, FITC-conjugated; Sigma) and ALB (goat, FITC-conjugated; Bethyl Laboratories Montgomery, TX). HDAC inhibition was assessed using anti-acetyl histone H4 antibody (rabbit, Upstate Biotechnology, NY, USA). Respective secondary antibodies came from Jackson Immunoresearch, Cambridgeshire, UK. As a negative control, cells were incubated with appropriate gamma immunoglobulines (Jackson Immunoresearch) and immunostained under the same conditions. Cells were analysed using fluorescence microscopy with a Zeiss Axiovert scope.
Cell morphology was analysed using phase-contrast light-microscopy (Nikon).
ALB concentrations, secreted into the culture media, were analysed by ELISA .
Urea concentrations produced, after 24 hours-exposure of the cells to 6 mM NH4Cl (Sigma), were measured colometrically in the culture media according to the manufacturer's instructions (Quantichrom Urea assay kit, Bioassay Systems, Brussels, Belgium). Fresh culture media supplemented with 6 mM NH4Cl (Sigma) and 4 hours-cultured adult hepatocytes were used as negative and positive controls, respectively.
EROD- and PROD-activities were assessed as previously described  with some minor modifications: in our set-up, cells were incubated with 20 μM 7-ethoxyresorufin and 18 μM 7-pentoxyresorufin (all from Sigma) for 3 hours and subsequently for 30 min with β-glucuronidase/arylsulfatase (Roche Applied Science, Vilvoorde, Belgium).
To evaluate the inducibility of CYP2B6 and CYP1A1/2, cells were, after 18 days of differentiation, exposed to PB (final concentration 1 mM) and MC (final concentration 2 μM; all from Sigma), respectively. Media, supplemented with either PB or MC, were daily renewed. Fresh culture media and 4 hours-cultured adult hepatocytes were used as negative and positive controls, respectively.
Results are expressed as mean ± SD. Statistical analyses were performed using Oneway Anova and Student's t-test. The significance level was set at 0.05.
Research was funded by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) and grants from the Research Council (OZR) of the Vrije Universiteit Brussel and Fund for Scientific Research (FWO-Vlaanderen), Belgium.
The authors thank Prof. Y. Vander Heyden and B. Dejaegher for their valuable statistical advice; Prof. L Bouwens and L. Baeyens for their dedicated scientific support and G. De Pauw, E. Desmedt, B. Degreef, H. Mertens and S. Coppens for their excellent technical assistance.
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