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Biomaterials 27 (2006) 40874097 www.elsevier.com/locate/biomaterials

Inducing hepatic differentiation of human mesenchymal stem cells in pellet culture

Shin-Yeu Onga, Hui Daia, Kam W. Leonga,b,
a

Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA b Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA Received 30 January 2006; accepted 15 March 2006

Abstract Extensive cellcell or cellmatrix interaction in three-dimensional (3D) culture is important for the maintenance of adult hepatocyte function and the maturation of hepatic progenitors. However, although there is signicant interest in inducing the transdifferentiation of adult stem cells into the hepatic lineage, very few studies have been conducted in a 3D culture conguration. The aim of this study is to investigate the differentiation of mesenchymal stem cells (MSC) into hepatocytes in a pellet conguration, with or without the presence of small intestinal submucosa (SIS). After 4 weeks of differentiation with growth factors bFGF, HGF, and OsM, we obtained hepatocyte-like cells that expressed a subset of hepatic genes, secreted albumin and urea, stored glycogen, and showed inducible CYP3A4 mRNA levels. When these cells were implanted into livers of hepatectomized rats, they secreted human albumin into the bloodstream. The hepatic differentiation of MSC was faster in cell pellets without SIS. The plausible explanations for this nding may be related to the mass transport issues of the two different pellets and the role of cellcell contact over cellmatrix interactions. The ndings of this study should help in the design of optimal culture congurations for efcient hepatic differentiation of adult stem cells. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Stem cell; Hepatocyte; Cell adhesion; ECM

1. Introduction The study of hepatic differentiation of mesenchymal stem cells (MSC) is important from both the clinical application and basic science perspectives. Not only is there an urgent need for an adequate supply of human hepatocytes for transplantation or for articial liver devices [1], the identication of molecular signals that underlie transdifferentiation advances understanding in developmental biology [2]. MSC-based therapy of the liver is attractive because autologous bone marrow-derived MSC can be harvested, expanded extensively ex vivo, and differentiated into a hepatic phenotype for transplantation back into the patient. The challenge remains to develop

Corresponding author. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. E-mail address: kam.leong@duke.edu (K.W. Leong).

robust protocols to generate hepatocytes from bone marrow-derived MSC for the treatment of liver disease. Most studies for the hepatic transdifferentiation of adult stem cells have been carried out in monolayer culture. However, hepatocytes are known to better maintain their differentiated functions in three-dimensional (3D) multicellular aggregates, or spheroids, than in monolayer culture [3,4]. Extensive cellcell contact between hepatocytes grown in aggregates promotes the formation of gap junctions, tight junctions, and bile canaliculi that are important for stabilizing the hepatocyte phenotype [5,6]. Cells in spheroids also have a morphology and ultrastructure similar to those found in a native liver lobule [7]. It has also been demonstrated that an increased level of Ecadherin mediated cell adhesion between cultured hepatocytes induces higher levels of liver-specic functions [8]. Many studies have also highlighted the benet of Matrigel, a basement membrane extract from the EngelbrethHolmSwarm mouse sarcoma that serves as a

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.03.022

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4088 S.-Y. Ong et al. / Biomaterials 27 (2006) 40874097 polypropylene conical tubes containing 5 mL of MSCGM. To determine the optimal pellet formation conditions, pellets were formed under three different conditions: (1) cell/SIS mixture centrifuged immediately; (2) cell/ SIS mixture incubated for 1 or 2 h before centrifugation; (3) cell/SIS mixture incubated on a shaker (Thermolyne Rotomix type 50800) at 50 rpm, 37 1C, 5 percent CO2 for 1 or 2 h before centrifugation. To make cell pellets without SIS fragments, 2 105 MSCs were spun down in the same manner. All centrifugation was carried out at 560g for 5 min. After centrifugation, the cells were incubated at 37 1C and 5 percent CO2. Within 16 h, the centrifuged cells formed spherical pellets that did not adhere to the walls of the tube.

complex extracellular matrix (ECM), in prolonging the maintenance of adult hepatocyte functions and in promoting the maturation of hepatic progenitor cells. Differentiation of hepatic progenitor cells is most complete on 3D Matrigel gels [9,10], and liver-specic functions of adult hepatocytes are better maintained when they are plated on a combination of ECM molecules [1113]. To date, various coatings like bronectin, collagen, and Matrigel have been used to support the differentiation of bone marrow stem cells to hepatocytes [1416]. To our knowledge, the only study comparing the use of various ECM molecules for hepatic differentiation of bone marrow stem cells have concluded that Matrigel supports differentiation better than individual ECM components [14]. The aim of this study is to investigate the hepatic differentiation of MSCs in two types of 3D constructs: a cell pellet, and a cell pellet supplemented with ECM from the small intestinal submucosa (SIS). SIS is a biomaterial derived from the porcine small intestine. It consists primarily of collagen I, as well as trace amounts of collagen IV, bronectin, and laminin [17]. Since SIS contains a combination of ECM molecules in its native state and concentration, it has the potential to provide the appropriate biological signals to guide hepatic differentiation in vitro. In addition, SIS supports angiogenesis in vivo [18], which will be essential for survival of the transplanted cells. In this study, the MSC were cultured in two different constructs with growth factors and monitored for their hepatic differentiation. The transdifferentiated MSC expressed a subset of hepatic markers and proteins, possessed inducible P450 activity, secreted albumin and urea, and stored glycogen. In a pilot experiment to assess the engraftment potential of these transdifferentiated cells, implantation of the cultured pellets into the liver of hepatectomized rats produced measurable human albumin in serum for up to 2 weeks.
2. Materials and methods 2.1. Human mesenchymal stem cell maintenance

2.4. Cell viability and distribution in pellets

To determine cell viability in the pellets that formed overnight, pellets were stained with the Live/Dead kit (Molecular ProbesTM Invitrogen) for 30 min at 37 1C and examined using an Ultraview II confocal laser scanning microscope. Metabolic activity of cells in newly formed pellets was also assessed using the cell proliferation reagent WST-1 (Roche Molecular Biochemicals). WST-1 is a tetrazolium salt that is cleaved by metabolically active cells to a formazan dye. Absorbance of the dye was quantied at 450 nm using a spectrophotometer. To determine cell distribution within the pellet, pellets that formed overnight were xed with 10 percent formalin at 4 1C overnight before embedding and sectioning. Cell nuclei were stained with DAPI (Molecular ProbesTM Invitrogen), and sections were examined using a uorescence microscope.

2.5. Hepatic differentiation of MSCs in pellet culture

It was determined that shaking the SIS/cell mixture for 2 h before centrifuging yielded pellets with higher cell viability and more uniform cell distribution within the SIS matrix. Thus, subsequent cell pellets with SIS were made using this method. To induce hepatic differentiation, cells in pellets with or without SIS fragments were cultured in a basal medium with 50 ng/mL of hepatocyte growth factor (HGF) and 10 ng/mL basic broblast growth factor (bFGF) for 2 weeks, followed by 50 ng/mL Oncostatin M (OsM) for 2 weeks. The basal medium consisted of Iscoves Modied Dulbeccos Medium (IMDM, Gibco) with 10 percent FBS (Sigma, St. Louis, MO), 50 mg/mL Insulin, transferrin, selenium premix (ITS+, Becton Dickinson, NJ), 0.5 mM Dexamethasone (Sigma), 0.61 g/L Nicotinamide (Sigma), and 0.2 mM ascorbic acid-2-phosphate. Cell pellets were fed twice a week, and media was collected and stored at 80 1C for the measurement of urea and albumin concentrations. After 4 weeks of differentiation, some cell pellets were maintained for 48 h in the presence of 20 mM clotrimazole (Sigma) to induce cytochrome (CYP) P450 activity.

2.6. Real-time PCR and semi-quantitative PCR

Human MSC (Cambrex, Walkersville, MD) were expanded in a standard MSC growth medium (MSCGM; Cambrex). To minimize variability, only cells of the sixth passage were used for the hepatic differentiation studies. RNA was extracted from the cell pellets using the RNEasy kit (Qiagen, Valencia, CA) according to the manufacturers instructions. DNase I (Qiagen) digestion was performed to minimize the possibility of genomic DNA contamination. The Sensiscript RT kit (Qiagen) and oligo dT1218 primers (Invitrogen) were used to reverse transcribe total RNA to cDNA for real-time PCR. Real-time PCR was performed using the SYBRs Green PCR Master Mix (Applied Biosystems, Rotkreuz, Switzerland), and the ABI Prism 7900 Sequence Detection System (Applied Biosystems). cDNA samples, 1 mL in a 50 mL volume reaction, were analyzed for the gene of interest and for the reference gene b-actin. Each sample was run in triplicate. PCR was performed at 50 1C for 2 min and 95 1C for 10 min, followed by 40 cycles of denaturation at 95 1C for 15 s, and annealing/ extension at 55 1C for 1 min. After amplication is complete, dissociation analysis was performed to ensure that no primer dimers were formed in samples, and that the reaction was specic. Quantitation curves and curve tting of unknowns were performed by the ABI7900 Prism SDS software. Semi-quantitative PCR was performed with 0.2 mg of RNA per 25 mL reaction using the one-step RT-PCR kit from Qiagen. PCR cycling

2.2. Preparation of SIS fragments

Dry SIS cell culture sheets (Sigma) were weighed and hydrated with PBS. To prepare SIS fragments, the hydrated SIS sheets were pulverized using a mortar and pestle set (320 ml, Fisher Scientic) under sterile conditions. The resulting SIS fragments were washed with PBS, and stored at 4 1C as a suspension in PBS containing 1 percent Penicillin/ Streptomycin (Gibco). The SIS fragments were used within a month.

2.3. Pellet formation

To form cell pellets supplemented with SIS fragments, approximately 150 mg of dry SIS fragments were mixed with 2 105 MSCs in 15 mL

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S.-Y. Ong et al. / Biomaterials 27 (2006) 40874097 Table 1 Primers used for PCR Gene b-actin CK18 Albumin TO a1-AT HNF4a CYP3A4 Aggrecan Col II-A/B Sequence F: 50 -GGGCATGGGTCAGAAGGATT-30 R: 50 -GAGGCGTACAGGGATAGCAC-30 F: 50 -AATGGGAGGCATCCAGAACGAGAA-30 R: 50 -GGGCATTGTCCACAGTATTTGCGA-30 F: 50 -ACAGAATCCTTGGTGAACAGGCGA-30 R: 50 -TCAGCCTTGCAGCACTTCTCTACA-30 F: 50 -AGTCAAACC TCCGTGCTT-30 R: 50 -TCGGTGCATCCGAGAAACA-30 F: 50 -TCGCTACAGCCTTTGCAATG-30 R: 50 -TTGAGGGTACGGAGGAGTTCC-30 F: 50 -GGAACATATGGGAACCAACG-30 R: 50 -AACTTCCTGCTTGGTGATGG-30 F: 50 -TCACCCTGATGTCCAGCAGAAACT-30 R: 50 -TACTTTGGGTCACGGTGAAGAGCA-30 F: 50 -GCC TTG AGC AGT TCA CCT TC-30 R: 50 -CTC TTC TAC GGG GAC AGC AG-30 F: 50 -GTGGAGCAGCAAGAGCAAGGA-30 R: 50 -CTTGCCCCACTTACCAGTGTG-30 Product (bp) 302 227 256 390 142 215 228 372 344 TA (1C) 56 58 55 52 55 52 55 54 57 4089

conditions consisted of an initial 30 min reverse transcription step at 50 1C, a 15 min activation step at 95 1C, and 40 cycles of denaturation at 94 1C for 1 min, annealing at primer-specic temperatures for 1 min, and extension at 72 1C for 1 min. Final products were separated by electrophoresis on 2 percent agarose gels stained with ethidium bromide. A description of primers, size of products, and annealing temperatures is listed in Table 1.

Hateld, PA) for 15 min. Color was developed in lukewarm tap water. Samples were then counterstained with Harris hematoxylin (Pierce Biotechnology) and assessed under light microscope. Controls for PAS staining were incubated with 5 g/L amylase (A3176, Sigma) for 15 min at room temperature to digest the glycogen prior to PAS staining.

2.10. In vitro implantation 2.7. Urea assay

The concentration of urea in culture media was measured using the colorimetric assay (640-1, Sigma) according to the manufacturers instructions. Dilutions of a stock solution of urea (Sigma) were used to create a standard curve. Samples from three separate cultures were analyzed in triplicate for each condition. The absorbance of the basal medium was subtracted from the absorbance of each test sample to obtain the nal absorbance value for determining urea concentrations from the standard curve. Cell pellets that have been cultured for 28 days were implanted into livers of hepatectomized rats. All rats were immunosuppressed by intraperitoneal injection of Cyclosporin A (10 mg/kg/day) one day prior to implantation and extending to 14 days after implantation. The animal surgery conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996). Rats were anesthetized by inhalation of isourane, and laparotomized. Following 70 percent partial hepatectomy of the middle and left lobe, three pellets were implanted into the right lobe of each recipient rat and the implantation site marked. Fourteen days after implantation, rats were killed and the lobes containing the cell pellets excised. On days 7 and 14, serum samples were collected and stored at 80 1C until ELISA was performed.

2.8. Albumin ELISA

An ELISA Quantitation kit from Bethyl Labs (Montgomery, Tx) was used to measure albumin levels in culture medium. Samples from three separate cultures were analyzed in triplicate for each condition. The absorbance of the basal medium was subtracted from each samples absorbance, and albumin concentration was determined from a standard curve. An ELISA kit specic for human albumin was used according to the manufacturers instructions (Cygnus Technologies, Plainville, MA). Normal rat serum showed an absorbance at or below the zero standard provided by the manufacturer. The nal absorbance of the test samples was obtained after subtracting the zero absorbance, and used to determine albumin concentrations in the serum from the standard curve. Samples were run in duplicate.

2.11. Immunostaining and histology

Cultured cell pellets and rat liver slices containing the pellets were xed at 4 1C in 10 percent formalin overnight, washed, dehydrated in 20 percent sucrose, embedded in parafn, and sectioned. Antigen retrieval was performed for ve minutes in 10 mM sodium citrate, pH 6 at 90100 1C. Sections were stained with antibodies against human albumin (clone HAS11, 1:250, Sigma) and human cytokeratin 18 (clone RCK106, 1:100, Chemicon). Rat liver sections were also stained with an antibody against human alpha-synuclein (clone 4B12, 1:100, Abcam) to identify human cell nuclei. After antigen retrieval, tissue sections and pellet sections were blocked with hydrogen peroxide and a protein block (Universal BlockerTM Blocking Buffer in TBS, Pierce Biotechonology, Rockport, IL) before incubation with antibodies for 30 min. These antibodies showed no cross reactivity with rat liver. For each staining, a negative control was performed by omitting the primary antibodies from the staining protocol. The sections were then visualized using the Dako LSAB2 HRP kit specic for rat tissue and the Dako Liquid diaminobenzidine (DAB) SubstrateChromagen System kit according to the manufacturers instructions. The

2.9. Periodic acidSchiff (PAS) staining for glycogen

Cultured cell pellets were xed in 10 percent formalin overnight at 4 1C, embedded in parafn, and sectioned. Sections were permeabilized with 0.1 percent Triton X-100 (Sigma) for 15 min. For PAS staining, slides were rst oxidized in 1 percent periodic acid (Sigma) for ve minutes, rinsed, and then treated with Schiffs reagent (Electron Microscopy Sciences,

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4090 S.-Y. Ong et al. / Biomaterials 27 (2006) 40874097 sections were then counterstained with hematoxylin. Routine H&E staining was also performed.

2.12. Statistical analysis

The Students t-test was used to evaluate differences between groups. Statistical signicance was established at Po0:05.

3. Results 3.1. Cell viability and distribution in pellets Cell pellets with incorporated SIS fragments were formed either by immediate centrifugation, or by incubating with or without shaking for some time prior to centrifugation. Cell/SIS pellets were larger (23 mm) than

pellets without SIS (o1 mm). Newly formed cell pellets were stained by the Live/dead kit. Incubating the cell/SIS mixture with or without shaking before centrifugation led to cell pellets with mostly viable cells (Fig. 1A). There were some dead cells in pellets formed by immediate centrifugation of the cell/SIS mixture (Fig. 1B), but the largest number of dead cells were seen in pellets without SIS fragments (Fig. 1C). From the Live/dead stain, it was also apparent that cells in pellets without SIS were more cuboidal and rounded than cells in pellets with SIS. This showed that incorporated SIS fragments served as a scaffold for cell attachment and spreading. These observations were conrmed by the WST-1 assay. WST-1 metabolism was higher in cell pellets that were formed by centrifuging after incubating the cell/SIS mixture for some time with or without shaking (Fig. 1G). Lastly, cell

Fig. 1. Cell viability in pellets as assessed by Live/dead stain. Cells in pellets formed by centrifugation after shaking or incubating the cell/SIS mixture were mostly viable and stained green (A, representative image). There were some dead (red) cells in pellets formed by centrifuging the cell/SIS mixture immediately (B). Even more dead cells were visible in pellets without SIS (C). Sections of cell pellets stained with DAPI revealed that cells were more evenly distributed in pellets that were formed by shaking the cell/SIS mixture for 1 h before centrifuging (D), compared to those that were centrifuged immediately (E). Cell pellets without SIS had extensive cellcell contacts (F). WST-1 assay showed that in general, incubating the cell/SIS mixture with or without shaking before centrifugation led to higher cell metabolic activity (G). Po0:05; n 3. Scale bars represent 100 mm.

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distribution in pellets with SIS fragments were more uniform if shaking of the SIS/cell mixture was performed before centrifugation (Figs. 1D and E). Cell pellets without SIS fragments were also stained with DAPI (Fig. 1F).

3.3. Immunohistochemistry Albumin protein staining was visible from day 14 in both pellet congurations (Figs. 3A and D), and the intensity of staining increased with time of differentiation (Figs. 3B and E). In general, albumin protein was expressed in cells nearer the edge of the pellets before cells at the interior of the pellets. More intense staining for albumin protein was observed in pellets without SIS than in pellets with SIS. In addition, only cell pellets without SIS were stained for CK18 (Fig. 3F). Immunohistochemical staining of CK18 could not be observed in cell pellets with SIS despite detectable levels of CK18 mRNA by PCR (Fig. 3C). The omission of primary antibodies from the staining protocol resulted in negative staining in both pellets with SIS (Fig. 3G) and pellets without SIS (Fig. 3H). In 28-day cultured cell/SIS pellets, cell migration to the surface of the pellet can be observed (Fig. 3I). There were no observable changes in cell distribution in pellets without SIS throughout the culture period (data not shown).

3.2. Real-time PCR and semi-quantitative PCR PCR analysis showed that MSC differentiated in 3D constructs expressed a subset of hepatic genes, enzymes and transcription factors (Fig. 2A). Quantitative analysis of two late markers of hepatic differentiation, albumin and cytochrome P450 3A4 (CYP3A4), by real-time PCR was also performed (Figs. 2B and C). Albumin expression increased over the culture period, suggesting hepatic maturation. In addition, mRNA levels of CYP3A4 increased after addition of clotrimazole, a CYP inducer. This indicated that differentiated cells have inducible CYP activity, similar to hepatocytes. Based on PCR results, cells cultured as a spheroidal aggregate without SIS differentiated into hepatocytes more efciently. These pellets had signicantly higher levels of albumin and CYP3A4 mRNA during the differentiation period, expressed HNF4a transcription factor more readily, and induced higher levels of CYP3A upon exposure to clotrimazole.

3.4. Functional assays Albumin and urea synthesis, as well as glycogen production are unique to hepatocytes. MSC in 3D

Fig. 2. Expression of liver-specic genes. Liver-specic gene expression was induced by growth factors in MSCs cultured in a pellet conguration by 14 days (D14) and 28 days (D28) of differentiation (A). Quantitative analysis of albumin and CYP3A4 by real-time PCR was normalized to b-actin (B and C, respectively). D28 Clz: pellets that were cultured with clotrimazole for 2 days following the 28-day differentiation period to induce CYP3A expression; CK18: cytokeratin 18; TO: tryptophan 2,3-dioxygenase; a1-AT: a-1 antitrypsin; HNF4a: hepatocyte nuclear factor-4a; CYP3A4: cytochrome P450 3A4. Po0:001, n 3.

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Fig. 3. Immunohistochemical staining of albumin in cultured pellets. Positive staining of albumin was evidenced by DAB (brown) in pellets with SIS on day 14 (A) and day 28 (B). Staining for CK18 protein was not detectable in pellets with SIS at the end of the culture period (C). Intense staining for albumin was observed in cell pellets without SIS on day 14 (D) and day 28 (E). A few cells in pellets without SIS stained for CK18 on day 28 (F). Omission of the primary antibody from the staining protocol resulted in negative staining for both 28-day cultured cell pellets with SIS (G) and without SIS (H). Cell distribution in cell/SIS pellets after 4 weeks (I). Scale bars represent 100 mm.

constructs cultured in hepatic differentiation media gained albumin and urea synthetic capabilities (Figs. 4A and B). Albumin synthesis increased over the culture period, and MSC in pellets without SIS produced much higher levels of albumin compared to MSC in pellets with SIS. Urea production of cells in pellets without SIS was signicantly higher than in pellets with SIS by day 28. Glycogen staining was present in both cell pellets with and without SIS after 28 days of differentiation (Fig. 4 Ci, iii). Amylase digestion removed glycogen from the samples, resulting in negative PAS staining (Fig. 4 Cii, iv). 3.5. Hepatocyte-like cells in vivo Two weeks after transplantation of the pellets containing hepatocyte-like cells into rat livers, the tissue sections were analyzed for the expression of human albumin. Cells expressing human albumin were observed by immunohistochemistry in the livers of rats in which pellets with (Figs. 5B and C) or without (Figs. 5E and F) SIS were implanted. Corresponding serial sections were stained

with an antibody that localized to human cell nuclei, and conrmed that most human cells were positive for albumin (Figs. 5A and D). Rat hepatocytes were not stained by human albumin or human nuclei (Fig. 5G). Additionally, the implanted SIS/cell pellet recruited the hosts vasculature, and blood vessels penetrating the cell pellet with SIS were visible at day 7 (Fig. 5H) and day 14 (Fig. 5I). 3.6. Human albumin levels in rat serum Three pellets were implanted into the right liver lobe of each hepatectomized rat. To determine if the hepatocytelike cells transplanted in vivo secreted human albumin protein into the bloodstream, we performed an ELISA specic for human albumin. At the end of 2 weeks, human albumin was detectable in serum of rats transplanted with MSC transdifferentiated in both pellet congurations. The albumin levels measured in serum of rats that were transplanted with MSC differentiated in pellets without SIS was 0.1971.2 ng/mL on day 7 and 6.771.7 ng/mL on

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Fig. 4. Hepatocyte-specic functions of cells in cultured pellets. Albumin production, expressed as pg per 10,000 cells per hour, Po0:001, n 3 (A). Urea production, expressed as pg per cell per hour, Po0:05, n 3 (B). Differentiated MSCs produced and stored glycogen in both pellets with SIS (Ci) and pellets without SIS (Ciii). Specicity of staining was determined by digestion of glycogen prior to PAS staining (Cii, Civ), which eliminated the magenta staining for glycogen. Scale bars represent 100 mm.

day 14 (n 2). For the rat transplanted with MSC differentiated in pellets with SIS, human serum albumin was undetectable on day 7, but increased to a detectable level at week 20.35 ng/mL (n 1). 4. Discussion In this study we evaluated the hepatic differentiation of MSC in cell pellets, and cell pellets supplemented with ECM from the SIS. The hypothesis was that cellcell and cellmatrix interactions in a 3D environment, coupled with

appropriate chemical cues from growth factors, will aid in the hepatic transdifferentiation of adult stem cells. To our knowledge, one study examined the differentiation of HNF3b-transfected embryonic stem cells (ESC) in spheroidal aggregates [19], but no one has reported the hepatic differentiation of bone marrow MSC in a cell pellet conguration. Similarly, some studies induced hepatic differentiation of bone marrow stem cells and ESC in 3D collagen type I gels and scaffolds [20,21], but hepatic differentiation of bone marrow MSC on the SIS biomaterial has not been studied.

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Fig. 5. Pilot experiment to assess in vivo survival of transdifferentiated MSC. Transdifferentiated MSC were implanted into the liver of 70 percent hepatectomized rats. Two weeks post-implantation, sections were stained with antibodies specic for human albumin (B, C, E, F) and antibodies specic for human nuclei (A, D). Human cells in pellets with SIS (AC) and in pellets without SIS (DF) expressed albumin protein in rat livers. (C) and (F) are magnied images of the boxed areas in (B) and (E), respectively. Rat livers not implanted with pellets containing human cells did not stain with antibodies against human albumin or human nuclei (G). Implanted SIS/cell pellets recruited the host vasculature, and blood vessels penetrating the cell pellet with SIS were visible at day 7 (H) and day 14 (I). Scale bars represent 100 mm.

We found that hepatic differentiation of MSC in cell pellets was more efcient without than with SIS in the pellet. Hepatocyte-like cells in pellets without SIS expressed late markers of hepatocyte differentiation, albumin and CYP3A, at higher levels as quantied by real-time PCR. The expression of HNF4a, a transcription factor that regulates many liver genes, was also expressed earlier in cells differentiated in pellets without SIS. Cells in these pellets secreted more urea and albumin into the culture medium, and when implanted, these cells secreted more albumin into the bloodstream. Since a pellet culture of MSC has been used widely to mimic chondrogenic condensation in vitro [22], we also analyzed by PCR the expression of aggrecan and collagen II mRNA to determine if some MSC were differentiating into the chondrogenic lineage. Aggrecan was expressed in both pellet congurations after differentiation with HGF and bFGF, but was downregulated when OsM was applied after day 14. The levels of mRNA associated with chondrogenesis, aggrecan and collagen II, were lower or

undetectable in cell pellets without SIS, indicating that fewer cells in cell pellets without SIS were differentiating into the chondrogenic lineage. Less efcient differentiation in pellets containing SIS may be due to the following factors: TGFb, an important factor for chondrogenesis, is present in the SIS scaffold [23,24]. In a previous study of chondrogenic differentiation of Cambrex MSC, TGFb alone signicantly increases the gene expression of sox-9, aggrecan, and collagen II [25]. Since SIS is a naturally derived source of ECM that still retains bioactive growth factors after processing [24], as yet unidentied factors may interfere with the mechanisms of HGF, bFGF and OsM. Importantly, high-density pellet culture without SIS maximized cellcell contact, and cells in those pellets adopted a cuboidal morphology similar to hepatocytes. Cell density in the pellet with SIS may not be high enough for hepatic differentiation. Schwartz et al. [14] found that below 12.5 103 cells/cm2, no hepatic differentiation of human MSC was observed in monolayer on collagen, Matrigel, or bronectin. In

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addition, E-cadherin-expressing ESC matured into hepatocytes faster than cadherin-decient ESC, and exhibited better aggregation in response to growth factors that induce hepatic differentiation [26]. Presence of cellcell adhesion mediated by E-cadherins also increases the amount of HNF4a binding activity, leading to increased gene transcription [27]. Poorer differentiation in the pellets containing SIS suggests that cellcell interactions may play a more important role in inducing hepatic differentiation than cellmatrix interactions, at least in the case of SIS which might contain embedded growth factors that divert the differentiation of the MSC into other lineages. However, possible hypoxia in the cell/SIS pellets confounds the interpretation of the differentiation result. After a 28-day culture, cell density increased near the surface, and decreased at the interior of the cell/SIS pellet culture, suggesting hMSC migration to the pellet surface. This could be a response to overcome insufcient oxygen concentration at the center of the pellet [28] that is enhanced by the chemotactic effect of HGF [29]. The number of cells, and thus oxygen and nutrient requirements, in the pellets with or without SIS was the same. However, incorporated ECM led to a larger cell/SIS pellet compared to cell pellets without SIS. Malda et al. [30] found that oxygen tension in a 3D porous ber-deposited scaffold decreased from 21 percent at the scaffold surface to about 5 percent at a 2 mm depth after 14 days. Furthermore, the large ECM molecules constituting the SIS biomaterial may have hindered intra-pellet diffusion, leading to a more pronounced oxygen and nutrient gradient in the pellet, and an intensied hypoxic environment at the center. Oxygen tension has been shown to play an important role in MSC cultures. MSC expanded under an atmosphere of low oxygen (2 percent), representative of in vivo oxygen tension in the bone marrow, expressed higher levels of stem cell genes (Oct-4, Rex-1) than cells cultured at 20 percent oxygen [31], similar to ndings that ESC had reduced spontaneous cell differentiation in hypoxic environments (35 percent oxygen) [32]. Upon differentiation, MSC expanded under hypoxic conditions (25 percent) also expressed higher levels of osteoblastic and adipocytic markers than normoxic (20 percent) controls [31,33]. Hypoxia also activates Sox-9, a key transcription factor required for chondrogenesis in bone marrow stromal cells [34]. On the one hand, a more hypoxic environment may have driven MSC differentiation into the chondrogenic lineage, explaining the higher expression of chondrogenic differentiation markers in the cell/SIS pellet. On the other hand, the association between stemness and hypoxia precludes us from denitively concluding that hypoxia is adversely affecting the hepatic transdifferentiation of hMSC. Immunohistochemical analysis of albumin indicated that cells at the edge of cell/SIS pellet differentiated earlier than cells in the interior, suggesting that diffusional limitation of oxygen and nutrients adversely affected hepatic differentiation. However, hypoxic cells that mi-

grated to the edge of the pellet also re-established cellcell contacts with other cells at the edge of the pellet, and were further from the inuence of embedded growth factors in the SIS biomaterial. Thus, the observation that cells at the edge of the pellet differentiated earlier than cells at the interior may be due to all these factors: sufcient access to growth factors and oxygen, cellcell contact, and noninterference from embedded growth factors in the SIS. Despite the confounding factors precluding us from unraveling the underlying mechanisms of poorer differentiation in the cell/SIS pellet, the hepatocyte-like cells generated by our methods expressed a subset of hepatic markers and proteins, possessed inducible P450 activity, secreted albumin and urea, and stored glycogen. Preliminary analysis of differentiation with lower concentrations of HGF and OsM at 20 ng/mL in serum-free media led to poor differentiation of the cells. Thus, we increased the dose of HGF and OsM to 50 ng/mL, which has been shown to be effective for hepatic differentiation of umbilical cord blood derived-MSC [35]. Despite this, albumin secretion of cells differentiated in pellets without SIS for 4 weeks were still about 35 times lower than mature human hepatocytes [36]. Albumin and CYP3A4 mRNA levels of cells differentiated in pellets without SIS were also about 30 and 8 times lower respectively, than the HepG2 hepatocyte cell line [37]. Nonetheless, it is encouraging that these cells when transplanted into the rat liver could produce albumin into the bloodstream at measurable levels. Better design of the cell pellet with incorporated ECM by either making it smaller or incorporating pores to improve mass transport to cells at the center of the pellet may improve hepatic transdifferentiation. Instead of using SIS, the use of a liverderived biomatrix [38,39], which represents a physiological microenvironment for hepatocytes, may incorporate more appropriate factors to guide differentiation to the hepatic lineage. Since primary hepatocytes have limited capacity to expand ex vivo [40], the possibility of obtaining functionally comparable hepatocyte-like cells from MSC may alleviate cell shortage problems. Allogeneic MSC-based products have the advantage over autologous MSC in not requiring harvesting, expansion, and differentiation of cells from every patient requiring treatment. However, the immune responses leading to allogeneic MSC rejection need to be characterized and managed before these cells can be applied clinically. 5. Conclusion In summary, 3D spheroidal cultures are promising congurations for hepatic transdifferentiation of MSC. 3D pellet culture allows stable cell anchorage, permits the retention of ECM molecules produced by the cells, and as high-density cultures can be readily implanted into the liver or used in bioarticial liver devices. Possible confounding factors of diffusion limitation preclude us from concluding whether the SIS biomaterial is advantageous for hepatic

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differentiation. Insights gained from this study will be useful for designing optimal culture systems for the hepatic transdifferentiation of MSC. Acknowledgment This project is partially supported by the Division of Johns Hopkins in Singapore through a grant by A*STAR of Singapore, and a scholarship from DSTA of Singapore to SYO. We would like to thank Ms. Leena Kadakia for sharing the protocol for making the MSC/SIS pellets. References
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