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Biomaterials 27 (2006) 4087–4097

www.elsevier.com/locate/biomaterials

Inducing hepatic differentiation of human mesenchymalstem cells in pellet culture

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

aDepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USAbDepartment of Biomedical Engineering, Duke University, Durham, NC 27708, USA

Received 30 January 2006; accepted 15 March 2006

Abstract

Extensive cell–cell or cell–matrix 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 significant interest in inducing the transdifferentiation of

adult stem cells into the hepatic lineage, very few studies have been conducted in a 3D culture configuration. The aim of this study is to

investigate the differentiation of mesenchymal stem cells (MSC) into hepatocytes in a pellet configuration, 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 finding may be related to the

mass transport issues of the two different pellets and the role of cell–cell contact over cell–matrix interactions. The findings of this study

should help in the design of optimal culture configurations for efficient 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 mesenchymalstem cells (MSC) is important from both the clinicalapplication and basic science perspectives. Not only is therean urgent need for an adequate supply of humanhepatocytes for transplantation or for artificial liver devices[1], the identification of molecular signals that underlietransdifferentiation advances understanding in develop-mental biology [2]. MSC-based therapy of the liver isattractive because autologous bone marrow-derived MSCcan be harvested, expanded extensively ex vivo, anddifferentiated into a hepatic phenotype for transplantationback into the patient. The challenge remains to develop

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.03.022

ing author. Department of Biomedical Engineering, Johns

rsity School of Medicine, Baltimore, MD 21205, USA.

ess: kam.leong@duke.edu (K.W. Leong).

robust protocols to generate hepatocytes from bonemarrow-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 theirdifferentiated functions in three-dimensional (3D) multi-cellular aggregates, or spheroids, than in monolayer culture[3,4]. Extensive cell–cell contact between hepatocytesgrown in aggregates promotes the formation of gapjunctions, tight junctions, and bile canaliculi that areimportant for stabilizing the hepatocyte phenotype [5,6].Cells in spheroids also have a morphology and ultra-structure similar to those found in a native liver lobule [7].It has also been demonstrated that an increased level of E-cadherin mediated cell adhesion between cultured hepato-cytes induces higher levels of liver-specific functions [8].Many studies have also highlighted the benefit of

Matrigel, a basement membrane extract from the Engel-breth–Holm–Swarm mouse sarcoma that serves as a

ARTICLE IN PRESSS.-Y. Ong et al. / Biomaterials 27 (2006) 4087–40974088

complex extracellular matrix (ECM), in prolonging themaintenance of adult hepatocyte functions and in promot-ing the maturation of hepatic progenitor cells. Differentia-tion of hepatic progenitor cells is most complete on 3DMatrigel gels [9,10], and liver-specific functions of adulthepatocytes are better maintained when they are plated ona combination of ECM molecules [11–13]. To date, variouscoatings like fibronectin, collagen, and Matrigel have beenused to support the differentiation of bone marrow stemcells to hepatocytes [14–16]. To our knowledge, the onlystudy comparing the use of various ECM molecules forhepatic differentiation of bone marrow stem cells haveconcluded that Matrigel supports differentiation betterthan individual ECM components [14].

The aim of this study is to investigate the hepaticdifferentiation of MSCs in two types of 3D constructs: acell pellet, and a cell pellet supplemented with ECM fromthe small intestinal submucosa (SIS). SIS is a biomaterialderived from the porcine small intestine. It consistsprimarily of collagen I, as well as trace amounts ofcollagen IV, fibronectin, and laminin [17]. Since SIScontains a combination of ECM molecules in its nativestate and concentration, it has the potential to provide theappropriate biological signals to guide hepatic differentia-tion in vitro. In addition, SIS supports angiogenesis in vivo[18], which will be essential for survival of the transplantedcells. In this study, the MSC were cultured in two differentconstructs with growth factors and monitored for theirhepatic differentiation. The transdifferentiated MSC ex-pressed a subset of hepatic markers and proteins, possessedinducible P450 activity, secreted albumin and urea, andstored glycogen. In a pilot experiment to assess theengraftment potential of these transdifferentiated cells,implantation of the cultured pellets into the liver ofhepatectomized rats produced measurable human albuminin serum for up to 2 weeks.

2. Materials and methods

2.1. Human mesenchymal stem cell maintenance

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.

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 (320ml, Fisher Scientific) 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

150mg of dry SIS fragments were mixed with 2� 105 MSCs in 15mL

polypropylene conical tubes containing 5mL 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 5min. 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.

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

30min 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

quantified at 450 nm using a spectrophotometer. To determine cell

distribution within the pellet, pellets that formed overnight were fixed

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 fluorescence 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

fibroblast growth factor (bFGF) for 2 weeks, followed by 50 ng/mL

Oncostatin M (OsM) for 2 weeks. The basal medium consisted of Iscove’s

Modified Dulbecco’s Medium (IMDM, Gibco) with 10 percent FBS

(Sigma, St. Louis, MO), 50mg/mL Insulin, transferrin, selenium premix

(ITS+, Becton Dickinson, NJ), 0.5 mM Dexamethasone (Sigma), 0.61 g/L

Nicotinamide (Sigma), and 0.2mM 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

RNA was extracted from the cell pellets using the RNEasy kit (Qiagen,

Valencia, CA) according to the manufacturer’s instructions. DNase I

(Qiagen) digestion was performed to minimize the possibility of genomic

DNA contamination. The Sensiscript RT kit (Qiagen) and oligo dT12–18

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, 1mL in a 50mL 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 2min and 95 1C for 10min,

followed by 40 cycles of denaturation at 95 1C for 15 s, and annealing/

extension at 55 1C for 1min. After amplification is complete, dissociation

analysis was performed to ensure that no primer dimers were formed in

samples, and that the reaction was specific. Quantitation curves and curve

fitting of unknowns were performed by the ABI7900 Prism SDS software.

Semi-quantitative PCR was performed with 0.2 mg of RNA per 25mLreaction using the one-step RT-PCR kit from Qiagen. PCR cycling

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Table 1

Primers used for PCR

Gene Sequence Product (bp) TA (1C)

b-actin F: 50-GGGCATGGGTCAGAAGGATT-30 302 56

R: 50-GAGGCGTACAGGGATAGCAC-30

CK18 F: 50-AATGGGAGGCATCCAGAACGAGAA-30 227 58

R: 50-GGGCATTGTCCACAGTATTTGCGA-30

Albumin F: 50-ACAGAATCCTTGGTGAACAGGCGA-30 256 55

R: 50-TCAGCCTTGCAGCACTTCTCTACA-30

TO F: 50-AGTCAAACC TCCGTGCTT-30 390 52

R: 50-TCGGTGCATCCGAGAAACA-30

a1-AT F: 50-TCGCTACAGCCTTTGCAATG-30 142 55

R: 50-TTGAGGGTACGGAGGAGTTCC-30

HNF4a F: 50-GGAACATATGGGAACCAACG-30 215 52

R: 50-AACTTCCTGCTTGGTGATGG-30

CYP3A4 F: 50-TCACCCTGATGTCCAGCAGAAACT-30 228 55

R: 50-TACTTTGGGTCACGGTGAAGAGCA-30

Aggrecan F: 50-GCC TTG AGC AGT TCA CCT TC-30 372 54

R: 50-CTC TTC TAC GGG GAC AGC AG-30

Col II-A/B F: 50-GTGGAGCAGCAAGAGCAAGGA-30 344 57

R: 50-CTTGCCCCACTTACCAGTGTG-30

S.-Y. Ong et al. / Biomaterials 27 (2006) 4087–4097 4089

conditions consisted of an initial 30min reverse transcription step at 50 1C,

a 15min activation step at 95 1C, and 40 cycles of denaturation at 94 1C for

1min, annealing at primer-specific temperatures for 1min, and extension

at 72 1C for 1min. 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.

2.7. Urea assay

The concentration of urea in culture media was measured using the

colorimetric assay (640-1, Sigma) according to the manufacturer’s

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 final absorbance value for determining urea concentrations from the

standard curve.

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 sample’s

absorbance, and albumin concentration was determined from a standard

curve. An ELISA kit specific for human albumin was used according to

the manufacturer’s instructions (Cygnus Technologies, Plainville, MA).

Normal rat serum showed an absorbance at or below the zero standard

provided by the manufacturer. The final 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.

2.9. Periodic acid–Schiff (PAS) staining for glycogen

Cultured cell pellets were fixed in 10 percent formalin overnight at 4 1C,

embedded in paraffin, and sectioned. Sections were permeabilized with 0.1

percent Triton X-100 (Sigma) for 15min. For PAS staining, slides were

first oxidized in 1 percent periodic acid (Sigma) for five minutes, rinsed,

and then treated with Schiff’s reagent (Electron Microscopy Sciences,

Hatfield, PA) for 15min. 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 15min at

room temperature to digest the glycogen prior to PAS staining.

2.10. In vitro implantation

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 (10mg/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 isoflurane, and laparotomized. Following 70 percent partial hepatect-

omy 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.

2.11. Immunostaining and histology

Cultured cell pellets and rat liver slices containing the pellets were fixed

at 4 1C in 10 percent formalin overnight, washed, dehydrated in 20 percent

sucrose, embedded in paraffin, and sectioned. Antigen retrieval was

performed for five minutes in 10mM sodium citrate, pH 6 at 90–100 1C.

Sections were stained with antibodies against human albumin (clone HAS-

11, 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 30min. 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 specific

for rat tissue and the Dako Liquid diaminobenzidine (DAB) Substrate-

Chromagen System kit according to the manufacturer’s instructions. The

ARTICLE IN PRESSS.-Y. Ong et al. / Biomaterials 27 (2006) 4087–40974090

sections were then counterstained with hematoxylin. Routine H&E

staining was also performed.

2.12. Statistical analysis

The Student’s t-test was used to evaluate differences between groups.

Statistical significance was established at Po0:05.

3. Results

3.1. Cell viability and distribution in pellets

Cell pellets with incorporated SIS fragments wereformed either by immediate centrifugation, or by incubat-ing with or without shaking for some time prior tocentrifugation. Cell/SIS pellets were larger (2–3mm) than

Fig. 1. Cell viability in pellets as assessed by Live/dead stain. Cells in pellets for

mostly viable and stained green (A, representative image). There were som

immediately (B). Even more dead cells were visible in pellets without SIS (C). Se

distributed in pellets that were formed by shaking the cell/SIS mixture for

immediately (E). Cell pellets without SIS had extensive cell–cell contacts (F). W

without shaking before centrifugation led to higher cell metabolic activity (G)

pellets without SIS (o1mm). Newly formed cell pelletswere stained by the Live/dead kit. Incubating the cell/SISmixture with or without shaking before centrifugationled to cell pellets with mostly viable cells (Fig. 1A). Therewere some dead cells in pellets formed by immediatecentrifugation of the cell/SIS mixture (Fig. 1B), but thelargest number of dead cells were seen in pellets withoutSIS fragments (Fig. 1C). From the Live/dead stain, it wasalso apparent that cells in pellets without SIS were morecuboidal and rounded than cells in pellets with SIS. Thisshowed that incorporated SIS fragments served as ascaffold for cell attachment and spreading. These observa-tions were confirmed by the WST-1 assay. WST-1metabolism was higher in cell pellets that were formedby centrifuging after incubating the cell/SIS mixture forsome time with or without shaking (Fig. 1G). Lastly, cell

med by centrifugation after shaking or incubating the cell/SIS mixture were

e dead (red) cells in pellets formed by centrifuging the cell/SIS mixture

ctions of cell pellets stained with DAPI revealed that cells were more evenly

1 h before centrifuging (D), compared to those that were centrifuged

ST-1 assay showed that in general, incubating the cell/SIS mixture with or

. �Po0:05; n ¼ 3. Scale bars represent 100mm.

ARTICLE IN PRESSS.-Y. Ong et al. / Biomaterials 27 (2006) 4087–4097 4091

distribution in pellets with SIS fragments were moreuniform if shaking of the SIS/cell mixture was per-formed before centrifugation (Figs. 1D and E). Cellpellets without SIS fragments were also stained with DAPI(Fig. 1F).

3.2. Real-time PCR and semi-quantitative PCR

PCR analysis showed that MSC differentiated in 3Dconstructs expressed a subset of hepatic genes, enzymesand transcription factors (Fig. 2A). Quantitative analysisof two late markers of hepatic differentiation, albumin andcytochrome P450 3A4 (CYP3A4), by real-time PCR wasalso performed (Figs. 2B and C). Albumin expressionincreased over the culture period, suggesting hepaticmaturation. In addition, mRNA levels of CYP3A4increased after addition of clotrimazole, a CYP inducer.This indicated that differentiated cells have inducible CYPactivity, similar to hepatocytes. Based on PCR results, cellscultured as a spheroidal aggregate without SIS differen-tiated into hepatocytes more efficiently. These pellets hadsignificantly higher levels of albumin and CYP3A4 mRNAduring the differentiation period, expressed HNF4a tran-scription factor more readily, and induced higher levels ofCYP3A upon exposure to clotrimazole.

Fig. 2. Expression of liver-specific genes. Liver-specific gene expression was in

days (D14) and 28 days (D28) of differentiation (A). Quantitative analysis of alb

respectively). D28 Clz: pellets that were cultured with clotrimazole for 2 days

CK18: cytokeratin 18; TO: tryptophan 2,3-dioxygenase; a1-AT: a-1 antitrypsin��Po0:001, n ¼ 3.

3.3. Immunohistochemistry

Albumin protein staining was visible from day 14 in bothpellet configurations (Figs. 3A and D), and the intensity ofstaining increased with time of differentiation (Figs. 3B andE). In general, albumin protein was expressed in cellsnearer the edge of the pellets before cells at the interior ofthe pellets. More intense staining for albumin protein wasobserved in pellets without SIS than in pellets with SIS. Inaddition, only cell pellets without SIS were stained forCK18 (Fig. 3F). Immunohistochemical staining of CK18could not be observed in cell pellets with SIS despitedetectable levels of CK18 mRNA by PCR (Fig. 3C). Theomission of primary antibodies from the staining protocolresulted in negative staining in both pellets with SIS (Fig.3G) and pellets without SIS (Fig. 3H). In 28-day culturedcell/SIS pellets, cell migration to the surface of the pelletcan be observed (Fig. 3I). There were no observablechanges in cell distribution in pellets without SIS through-out the culture period (data not shown).

3.4. Functional assays

Albumin and urea synthesis, as well as glycogenproduction are unique to hepatocytes. MSC in 3D

duced by growth factors in MSCs cultured in a pellet configuration by 14

umin and CYP3A4 by real-time PCR was normalized to b-actin (B and C,

following the 28-day differentiation period to induce CYP3A expression;

; HNF4a: hepatocyte nuclear factor-4a; CYP3A4: cytochrome P450 3A4.

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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 100mm.

S.-Y. Ong et al. / Biomaterials 27 (2006) 4087–40974092

constructs cultured in hepatic differentiation media gainedalbumin and urea synthetic capabilities (Figs. 4A and B).Albumin synthesis increased over the culture period, andMSC in pellets without SIS produced much higher levels ofalbumin compared to MSC in pellets with SIS. Ureaproduction of cells in pellets without SIS was significantlyhigher than in pellets with SIS by day 28. Glycogenstaining was present in both cell pellets with and withoutSIS after 28 days of differentiation (Fig. 4 Ci, iii). Amylasedigestion removed glycogen from the samples, resulting innegative PAS staining (Fig. 4 Cii, iv).

3.5. Hepatocyte-like cells in vivo

Two weeks after transplantation of the pellets contain-ing hepatocyte-like cells into rat livers, the tissue sectionswere analyzed for the expression of human albumin.Cells expressing human albumin were observed by im-munohistochemistry in the livers of rats in which pelletswith (Figs. 5B and C) or without (Figs. 5E and F) SISwere implanted. Corresponding serial sections were stained

with an antibody that localized to human cell nuclei,and confirmed that most human cells were positive foralbumin (Figs. 5A and D). Rat hepatocytes were notstained by human albumin or human nuclei (Fig. 5G).Additionally, the implanted SIS/cell pellet recruited thehosts’ vasculature, and blood vessels penetrating the cellpellet 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 ofeach hepatectomized rat. To determine if the hepatocyte-like cells transplanted in vivo secreted human albuminprotein into the bloodstream, we performed an ELISAspecific for human albumin. At the end of 2 weeks, humanalbumin was detectable in serum of rats transplanted withMSC transdifferentiated in both pellet configurations. Thealbumin levels measured in serum of rats that weretransplanted with MSC differentiated in pellets withoutSIS was 0.1971.2 ng/mL on day 7 and 6.771.7 ng/mL on

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Fig. 4. Hepatocyte-specific functions of cells in cultured pellets. Albumin production, expressed as pg per 10,000 cells per hour, ��Po0:001, n ¼ 3 (A).

Urea production, expressed as pg per cell per hour, �Po0:05, n ¼ 3 (B). Differentiated MSCs produced and stored glycogen in both pellets with SIS (Ci)

and pellets without SIS (Ciii). Specificity of staining was determined by digestion of glycogen prior to PAS staining (Cii, Civ), which eliminated the

magenta staining for glycogen. Scale bars represent 100mm.

S.-Y. Ong et al. / Biomaterials 27 (2006) 4087–4097 4093

day 14 (n ¼ 2). For the rat transplanted with MSCdifferentiated in pellets with SIS, human serum albuminwas undetectable on day 7, but increased to a detectablelevel at week 2–0.35 ng/mL (n ¼ 1).

4. Discussion

In this study we evaluated the hepatic differentiation ofMSC in cell pellets, and cell pellets supplemented withECM from the SIS. The hypothesis was that cell–cell andcell–matrix interactions in a 3D environment, coupled with

appropriate chemical cues from growth factors, will aid inthe hepatic transdifferentiation of adult stem cells. To ourknowledge, one study examined the differentiation ofHNF3b-transfected embryonic stem cells (ESC) in spher-oidal aggregates [19], but no one has reported the hepaticdifferentiation of bone marrow MSC in a cell pelletconfiguration. Similarly, some studies induced hepaticdifferentiation of bone marrow stem cells and ESC in 3Dcollagen type I gels and scaffolds [20,21], but hepaticdifferentiation of bone marrow MSC on the SIS biomater-ial 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 specific for human albumin (B, C, E, F) and antibodies specific

for human nuclei (A, D). Human cells in pellets with SIS (A–C) and in pellets without SIS (D–F) expressed albumin protein in rat livers. (C) and (F) are

magnified 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 100mm.

S.-Y. Ong et al. / Biomaterials 27 (2006) 4087–40974094

We found that hepatic differentiation of MSC in cellpellets was more efficient without than with SIS in thepellet. Hepatocyte-like cells in pellets without SIS ex-pressed late markers of hepatocyte differentiation, albuminand CYP3A, at higher levels as quantified by real-timePCR. The expression of HNF4a, a transcription factor thatregulates many liver genes, was also expressed earlier incells differentiated in pellets without SIS. Cells in thesepellets secreted more urea and albumin into the culturemedium, and when implanted, these cells secreted morealbumin into the bloodstream. Since a pellet culture ofMSC has been used widely to mimic chondrogeniccondensation in vitro [22], we also analyzed by PCR theexpression of aggrecan and collagen II mRNA todetermine if some MSC were differentiating into thechondrogenic lineage. Aggrecan was expressed in bothpellet configurations after differentiation with HGF andbFGF, but was downregulated when OsM was appliedafter day 14. The levels of mRNA associated withchondrogenesis, aggrecan and collagen II, were lower or

undetectable in cell pellets without SIS, indicating thatfewer cells in cell pellets without SIS were differentiatinginto the chondrogenic lineage.Less efficient differentiation in pellets containing SIS

may be due to the following factors: TGFb, an importantfactor for chondrogenesis, is present in the SIS scaffold[23,24]. In a previous study of chondrogenic differentiationof Cambrex MSC, TGFb alone significantly increases thegene expression of sox-9, aggrecan, and collagen II [25].Since SIS is a naturally derived source of ECM that stillretains bioactive growth factors after processing [24], as yetunidentified factors may interfere with the mechanisms ofHGF, bFGF and OsM. Importantly, high-density pelletculture without SIS maximized cell–cell contact, andcells in those pellets adopted a cuboidal morphologysimilar to hepatocytes. Cell density in the pellet with SISmay 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 inmonolayer on collagen, Matrigel, or fibronectin. In

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addition, E-cadherin-expressing ESC matured into hepa-tocytes faster than cadherin-deficient ESC, and exhibitedbetter aggregation in response to growth factors thatinduce hepatic differentiation [26]. Presence of cell–celladhesion mediated by E-cadherins also increases theamount of HNF4a binding activity, leading to increasedgene transcription [27]. Poorer differentiation in the pelletscontaining SIS suggests that cell–cell interactions may playa more important role in inducing hepatic differentiationthan cell–matrix interactions, at least in the case of SISwhich might contain embedded growth factors that divertthe differentiation of the MSC into other lineages.

However, possible hypoxia in the cell/SIS pellets con-founds the interpretation of the differentiation result. Aftera 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. Thiscould be a response to overcome insufficient oxygenconcentration at the center of the pellet [28] that isenhanced by the chemotactic effect of HGF [29]. Thenumber of cells, and thus oxygen and nutrient require-ments, in the pellets with or without SIS was the same.However, incorporated ECM led to a larger cell/SIS pelletcompared to cell pellets without SIS. Malda et al. [30]found that oxygen tension in a 3D porous fiber-depositedscaffold decreased from 21 percent at the scaffold surfaceto about 5 percent at a 2mm depth after 14 days.Furthermore, the large ECM molecules constituting theSIS biomaterial may have hindered intra-pellet diffusion,leading to a more pronounced oxygen and nutrientgradient in the pellet, and an intensified hypoxic environ-ment at the center.

Oxygen tension has been shown to play an importantrole in MSC cultures. MSC expanded under an atmosphereof low oxygen (2 percent), representative of in vivo oxygentension in the bone marrow, expressed higher levels of stemcell genes (Oct-4, Rex-1) than cells cultured at 20 percentoxygen [31], similar to findings that ESC had reducedspontaneous cell differentiation in hypoxic environments(3–5 percent oxygen) [32]. Upon differentiation, MSCexpanded under hypoxic conditions (2–5 percent) alsoexpressed higher levels of osteoblastic and adipocyticmarkers than normoxic (20 percent) controls [31,33].Hypoxia also activates Sox-9, a key transcription factorrequired for chondrogenesis in bone marrow stromal cells[34]. On the one hand, a more hypoxic environment mayhave driven MSC differentiation into the chondrogeniclineage, explaining the higher expression of chondrogenicdifferentiation markers in the cell/SIS pellet. On the otherhand, the association between stemness and hypoxiaprecludes us from definitively concluding that hypoxia isadversely affecting the hepatic transdifferentiation ofhMSC. Immunohistochemical analysis of albumin indi-cated that cells at the edge of cell/SIS pellet differentiatedearlier than cells in the interior, suggesting that diffusionallimitation of oxygen and nutrients adversely affectedhepatic differentiation. However, hypoxic cells that mi-

grated to the edge of the pellet also re-established cell–cellcontacts with other cells at the edge of the pellet, and werefurther from the influence of embedded growth factors inthe SIS biomaterial. Thus, the observation that cells at theedge of the pellet differentiated earlier than cells at theinterior may be due to all these factors: sufficient access togrowth factors and oxygen, cell–cell contact, and non-interference from embedded growth factors in the SIS.Despite the confounding factors precluding us from

unraveling the underlying mechanisms of poorer differ-entiation in the cell/SIS pellet, the hepatocyte-like cellsgenerated by our methods expressed a subset of hepaticmarkers and proteins, possessed inducible P450 activity,secreted albumin and urea, and stored glycogen. Prelimin-ary analysis of differentiation with lower concentrations ofHGF and OsM at 20 ng/mL in serum-free media led topoor differentiation of the cells. Thus, we increased thedose of HGF and OsM to 50 ng/mL, which has been shownto be effective for hepatic differentiation of umbilical cordblood derived-MSC [35]. Despite this, albumin secretion ofcells differentiated in pellets without SIS for 4 weeks werestill about 35 times lower than mature human hepatocytes[36]. Albumin and CYP3A4 mRNA levels of cellsdifferentiated in pellets without SIS were also about 30and 8 times lower respectively, than the HepG2 hepatocytecell line [37]. Nonetheless, it is encouraging that these cellswhen transplanted into the rat liver could produce albumininto the bloodstream at measurable levels. Better design ofthe cell pellet with incorporated ECM by either making itsmaller or incorporating pores to improve mass transportto cells at the center of the pellet may improve hepatictransdifferentiation. Instead of using SIS, the use of a liver-derived biomatrix [38,39], which represents a physiologicalmicroenvironment for hepatocytes, may incorporate moreappropriate factors to guide differentiation to the hepaticlineage.Since primary hepatocytes have limited capacity to

expand ex vivo [40], the possibility of obtaining function-ally comparable hepatocyte-like cells from MSC mayalleviate cell shortage problems. Allogeneic MSC-basedproducts have the advantage over autologous MSC in notrequiring harvesting, expansion, and differentiation of cellsfrom every patient requiring treatment. However, theimmune responses leading to allogeneic MSC rejectionneed to be characterized and managed before these cellscan be applied clinically.

5. Conclusion

In summary, 3D spheroidal cultures are promisingconfigurations for hepatic transdifferentiation of MSC.3D pellet culture allows stable cell anchorage, permits theretention of ECM molecules produced by the cells, and ashigh-density cultures can be readily implanted into the liveror used in bioartificial liver devices. Possible confoundingfactors of diffusion limitation preclude us from concludingwhether the SIS biomaterial is advantageous for hepatic

ARTICLE IN PRESSS.-Y. Ong et al. / Biomaterials 27 (2006) 4087–40974096

differentiation. Insights gained from this study will beuseful for designing optimal culture systems for the hepatictransdifferentiation of MSC.

Acknowledgment

This project is partially supported by the Division ofJohns Hopkins in Singapore through a grant by A*STARof Singapore, and a scholarship from DSTA of Singaporeto SYO. We would like to thank Ms. Leena Kadakia forsharing the protocol for making the MSC/SIS pellets.

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