Augmentation of EB-Directed Hepatocyte-Specific Function via Collagen

Sandwich and SNAP

Eric I. Novik, Jeffery Barminko, Tim J. Maguire, Nripen Sharma,Eric J. Wallenstein, and Rene S. SchlossDept. of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854

Martin L. YarmushDept. of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854

DOI 10.1021/bp.41Published online September 17, 2008 in Wiley InterScience (www.interscience.wiley.com).

The development of implantable engineered liver tissue constructs and ex vivo hepatocyte-based therapeutic devices are limited by an inadequate hepatocyte cell source. In our previousstudies, embryoid body (EB)-mediated stem cell differentiation spontaneously yielded popula-tions of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin(ALB) and cytokeratin-18 (CK18). However, these cultures neither yielded a homogenoushepatocyte lineage population nor exhibited detoxification function typical of a more maturehepatocyte lineage cell. In this study, secondary culture configurations were used to study theeffects of collagen sandwich culture and oncostatin-M (OSM) or S-nitroso-N-acetylpenicill-amine (SNAP) supplementation of EB-derived hepatocyte-lineage cell function. Quantitativeimmunofluorescence and secreted protein analyses were used to provide insights into thelong-term maintenance and augmentation of existing functions. The results of these studiessuggest that SNAP, independent of the collagen supplementation, maintained the highest levelsof ALB expression, however, mature liver-specific CK18 was only expressed in the presence ofgel sandwich culture supplemented with SNAP. In addition, albumin secretion and cytochromeP450 detoxification studies indicated that this condition was the best for the augmentation ofhepatocyte-like function. Maintenance and augmentation of hepatocyte-like cells isolated fromheterogeneous EB cell populations will be a critical step in generating large numbers of func-tional differentiated cells for therapeutic use.Keywords: ES cells, hepatocytes, collagen sandwich, SNAP, cytochrome P450

Introduction

Acute liver failure affects hundreds of thousands of peopleper year around the globe and in many cases is resolvedwith an orthotopic liver transplant. Because of the shortageof donor organs, many patients will die while waiting for adonor organ to be available. Extracorporeal liver assist devi-ces (LAD) could help to bridge patients for transplantanta-tion; however, this technology is limited by a lack of anadequate hepatocyte cell source (Tilles et al., 2002a,b). Pluri-potent embryonic stem (ES) cells represent a promisingrenewable cell source to generate hepatocyte lineage cells,which have been incorporated into implantable engineeredtissue constructs (Soto-Gutierrez et al., 2006) and ex vivocell-based therapeutic devices such as LADs (Cho et al.,2008). However, the current differentiation techniques havenot yet generated the large and functionally sustainable cellmasses, which would be required to make such therapiesclinically available.

ES differentiation into hepatocyte lineage cells, using avariety of differentiation platforms such as monolayer(Sharma et al., 2006), encapsulation (Maguire et al.,2006), and embryoid body (EB) mediated (Hamazakiet al., 2001; Heo et al., 2006; Kumashiro et al., 2005b),has been previously described by many investigators. Ofthese, EB-mediated differentiation, which mimics in vivoembryogenesis, has been characterized most completely.For example, following exogenous growth factor supple-mentation and coculture with nonparenchymal liver celllines, investigators have demonstrated that EB-mediateddifferentiation yields up to a 70% albumin (ALB)-positivepopulation, which expresses a variety of liver lineagegenes and metabolizes lidocane and diazepam (Soto-Gutierrez et al., 2007).

In addition, in vitro aggregation of murine ES cells ini-tiates the formation of EBs, which has been shown to facili-tate spontaneous differentiation in the absence of growthfactor and extracellular matrix supplementation, resulting inliver lineage cells characterized by 80% ALB expression aswell as mature hepatocyte genes such as cytochrome P450-detoxifying enzymes (CYP450) (Novik et al., 2006; Tsutsuiet al., 2006). However, despite the large number of studies

Correspondence concerning this article should be addressed to M. L.Yarmush at kma@soemail.rutgers.edu.

1132 Biotechnol. Prog. 2008, 24, 1132�1141

VVC 2008 American Institute of Chemical Engineers

reporting ES-hepatocyte lineage differentiation, there havebeen few reports of CYP450-related detoxification and drugmetabolism, which may be required for successful use ofthese cells for therapeutic treatment. Furthermore, mainte-nance of differentiated function and/or increased cell massafter the initial differentiation are critical steps for use ofES-generated in vitro liver lineage cells in a LAD or drugdiscovery studies. Finally, the selection of a homogenouscell population is essential to prevent undesirable cellularinteractions and functional expression. However, these issueshave been largely ignored. Previous studies investigated theeffects of oncostatin-M (OSM) and nitric oxide (NO) donorson fetal liver hepatocytes and have shown that their supple-mentation maintains long-term structure and function as wellas inducing further differentiation (Ehashi et al., 2005; Iwaiet al., 2002). In addition, studies have shown the potential ofcollagen sandwich cultures to augment hepatocyte popula-tions from within the heterogeneous populations (Depreteret al., 2000).

In this study, we have evaluated the effects of collagensandwich culture and S-nitroso-N-acetylpenicillamine (SNAP),a NO donor, or OSM supplementation on maintenance ofpreviously reported EB mediated spontaneously differenti-ated hepatocyte-lineage cell function. We have assessedexpression and secretion of ALB within cells secondarilycultured from the Day 17 EBs. In addition, using s-methyl-cholanthreene induction, we have assessed not only mainte-nance but also augmentation of function in the form ofCYP450-mediated detoxification. These studies identified asecondary culture condition, which maintained liver-likefunction initially observed after 17 days of spontaneousEB-mediated differentiation, and furthermore, promoted thedetoxification functions of Cyp450 enzymes.

Materials and Methods

Cell culture

All cell cultures were incubated in a humidified 378Cand 5% CO2 environment. The ES cell line D3 (ATCC,Manassas, VA) was maintained in an undifferentiated statein T-75 gelatin-coated flasks (Biocoat, BD-Biosciences,Bedford, MA) in Knockout Dulbecco’s modified Eagle’smedium (Gibco, Grand Island, NY) containing 15%knockout serum (Gibco), 4 mM L-glutamine (Gibco),100 U/mL penicillin (Gibco), 100 U/mL streptomycin(Gibco), 10 lg/mL gentamicin (Gibco), 1,000 U/mLESGROTM (Chemicon, Temecula, CA), 0.1 mM 2-mercap-toethanol (Sigma-Aldrich, St. Louis, MO). ESGRO con-tains leukemia inhibitory factor (LIF), which prevents EScell differentiation. Every 2 days, media was aspiratedand replaced with fresh media. Cultures were split andpassaged every 6 days, following media aspiration andwashing with 6 mL of phosphate-buffered solution (PBS)(Gibco). Cells were detached following incubation with3 mL of trypsin (Gibco) for 3 min, resulting in a singlecell suspension, and subsequently, the addition of 12 mLof knockout DMEM. Cells were then replated in gelatin-coated T-75 flasks at a density of 1 � 106 cells/mL. Stain-ing with Oct4, a recognized stem cell marker, demon-strated that the cells remained undifferentiated over theperiod used to accomplish these studies. 100% Oct4 stain-ing was observed at all passages (data not shown).

To induce differentiation, cells were suspended in Iscove’smodified Dulbecco’s medium (Gibco) containing 20% fetalbovine serum (Gibco), 4 mM L-glutamine (Gibco), 100 U/mL penicillin, 100 U/mL streptomycin (Gibco), and 10 lg/mL gentamicin (Gibco). EBs were formed and cultured for 2days using the hanging-drop method (1 � 103 ES cells per30 lL drop). The hanging drops where transferred to suspen-sion culture in 100-mm Petri dishes and cultured for an addi-tional 2 days. The EBs were then plated, one EB per well, in6-well tissue culture polystyrene plates (BD-Biosciences) foran additional 14 days. For secondary culture, Day-17 EBcells were detached following incubation with 5 mL of tryp-sin (Gibco) for 3 min, resulting in a single cell suspension,and subsequently, the addition of IMDM media. Cells fromDay-17 EBs were used because it has been observed that he-patocyte function is greatest on Day 17 (data not shown).Cells were then replated in 6-well tissue culture polystyrene(BD-Biosciences) at an initial seeding density of 5 � 104

Day 17 cells per well for further analysis. Culture mediumwas changed every 48 h. When OSM and SNAP were sup-plemented, 10 ng/mL OSM and 250 lM SNAP were addedto the culture medium. When collagen sandwich culture wasused, rat tail type I collagen (BD-Biosciences) gels were pre-pared by distributing 350 lL of collagen gel solution (3 parts1.33� DMEM, pH 7.4, and 1 part collagen solution at 4 mg/mL, chilled on ice and mixed immediately before use)evenly over one well of a 6-well plate (BD-Biosciences) andincubated at 378C for at least 1 h before use. A 5 � 105

cells were seeded in 2 mL of IMDM media on Day 0 and anadditional 350 lL of collagen gel solution was distributedover the cells after 1 day of culture. Therefore, the secondlayer of collagen is added on Day 1 of secondary cultureprotocol. One hour of incubation at 378C was allowed forgelation, and attachment of the second gel layer before themedium was replaced. Culture medium was changed every48 h.

The Hepa 1-6 cell line (ATCC, Manassas, VA) was main-tained in Dulbecco’s modified Eagle’s medium (Gibco) con-taining 10% fetal bovine serum (Gibco), 100 U/mLpenicillin (Gibco), 100 U/mL streptomycin (Gibco), and 4mM L-glutamine (Gibco). Hepa 1-6 cells were grown on tis-sue culture-treated T-75 flasks (Falcon, BD Biosciences, SanJose, CA). Hepa 1-6 cells were used as positive controls foreach of the following assays.

On evaluation days 4, 6, 8, and 10 days in secondary cul-ture, cells were replated into 12-well plates. Media sampleswere collected after 24 h of culture at 378C and 5% CO2.The cells were then washed in PBS (Gibco) and fixed in 4%paraformaldehyde (Sigma-Aldrich) in PBS for 15 min atroom temperature. Cells in collagen sandwich culture weredissociated with 0.5 mL of 0.1% collagenase (Sigma-Aldrich) in PBS for 30 min at 378C before replating into 12-well plates.

In situ indirect immunofluorescent cytokeratin-18 andintracellular albumin analysis

After 24 h in culture and fixing with 4% paraformalde-hyde, the cells were washed for 10 min in cold PBS andfixed in 4% paraformaldehyde (Sigma-Aldrich) in PBS for15 min at room temperature. The cells were washed twicefor 10 min in cold PBS and then twice for 10 min in coldsaponine (SAP)/PBS membrane permeabilization buffer con-taining 1% bovine serum albumin (BSA) (Sigma-Aldrich),

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0.5% SAP (Sigma-Aldrich), and 0.1% sodium azide (Sigma-Aldrich). To detect intracellular ALB, the cells were subse-quently incubated for 30 min at 48C in a SAP solution con-taining rabbit anti-mouse ALB antibody (150 lg/mL) (MPBiomedicals, Irvine, CA) or normal rabbit serum (150 lg/mL) (MP Biomedicals) as an isotype control, washed twicefor 10 min in cold SAP buffer, and then treated for 30 minat 48C with the secondary antibody, FITC-conjugated donkeyanti-rabbit, diluted 1:500 (Jackson Immuno Labs, Westgrove,PA). To detect cytokeratin-18 (CK18), which is produced inmature hepatocytes and a few other mature cell types, weincubated the cells for 30 min at 48C in a SAP solution con-taining rabbit anti-moue CK18 antibody (IgG1) (1:50 dilu-tion) (Santa Cruz Biotechnology) or the IgG1 fraction ofnormal rabbit serum (1:100 dilution) (Santa Cruz Biotechnol-ogy) as an isotype control, and then treated for 30 min at48C with the secondary antibody, FITC-conjugated goat anti-rabbit, diluted 1:200 (Jackson Immuno Labs, Westgrove,PA). For both stains, cells were then washed once with coldSAP buffer and once with cold PBS. Fluorescent imageswere acquired using a computer-interfaced inverted OlympusIX70 microscope. Specimens were excited using a 515-nmfilter. Fluorescent intensity values were determined for eachcell using Olympus Microsuite. Experimental intensity val-ues for each cell were calculated after subtracting the aver-age intensity of the isotype control.

Sandwich ELISA for detection of albumin secretion

To detect the secreted ALB within the media supernatantsobtained on each of the analysis days, we used a commer-cially available mouse ALB ELISA kit (Bethyl Laboratories,#E90-134). A standard curve was generated by creating se-rial dilutions of an ALB standard from 7.8 to 10,000 ng/mL.Absorbance readings were obtained using a Biorad (Hercu-les, CA) Model 680 plate reader with a 450-nm emissionfilter. ALB values were normalized to the cell numberrecorded on the day of media sample collection.

Urea secretion

Media samples were collected on all analysis days. Ureasynthesis was assayed using a commercially available kit(StanBio, Boerne, TX). A standard curve was generatedby creating serial dilutions of a urea standard from 0 to300 mg/mL. Absorbance readings were obtained usinga Biorad (Hercules, CA) Model 680 plate reader with a585-nm emission filter. Urea values were normalized tothe cell number recorded on the day of media samplecollection.

Measurement of cytochrome P450 activity

On evaluation days 4, 6, 8, and 10 days in secondary cul-ture, cells were replated into 12-well plates. 3-Methylcholan-threne was used at a concentration of 2 lM (Sigma–Aldrich)for 48 h before the addition of resorufin as an inducer of cyto-chrome P450 activities. Cytochrome P450-dependent benzy-loxyresorufin o-dealkylase activity (BROD, PROD, EROD,and MROD) was measured using resorufin substrates namelypentoxy-, benzyloxy-, ethoxy-, and methoxyresorufin from aResorufin Sampler Kit (Invitorgen, Carlsbad, CA). The incu-bation mixture contained resorufin substrates (pentoxy-,ethoxy-, or methoxyresorufin, final concentration 5 mM) anddicumarol (80 mM) in phenol red free Earle’s balanced salt

solution (EBSS) (Gibco). The prepared solutions were pre-heated to 378C before the incubation with cells. The 12-wellplates were washed with 2 mL of EBSS (378C) and furtherincubated with 2 mL of EBSS at 378C for 5–7 min to removethe residual medium. Following removal of EBSS, the incuba-tion mixture was added (2 mL per well) and the dishes wereincubated at 378C in a 5% CO2 incubator. At various timepoints (5, 10, 15, 20, 25 min) following incubation, 100 lL ofthe mixture was transferred into a 96-well plate. The fluores-cence of the plate was measured using a fluorescenceplate reader (DTX880, Beckman Coltour, Fullerton, CA,ext. 530 nm and emis. 590 nm) at the end of 25-min incu-bation. A standard curve of resorufin fluorescence was con-structed using concentrations ranging from 1 to 1,000 nmolin EBSS. A linear curve was obtained with an r2 of 0.99.The constructed standard curve was used to convert thefluorescence values obtained from the plate reader to nano-moles of resorufin. Rate of formation of resorufin, as calcu-lated from the early linear increase in the fluorescencecurve, was defined as cytochrome P450 activity andexpressed as nmol/min.

Statistical analysis of functional assays

Each data point represents the mean of three experiments(each with three biological replicates), and the error bars rep-resent the standard deviation of the mean. Statistical signifi-cance was determined using the Student’s t-test for unpaireddata. Differences were considered significant when the prob-ability was less then or equal to 0.05.

Results

Dynamic studies of secondarily cultured EB-derivedhepatocyte lineage cells

Our previous studies have demonstrated that spontaneousEB-mediated differentiation of ES cells yields a populationof cells displaying hepatocyte-specific characteristics such asALB and CK18 expression. However, these cultures did notyield a homogenous hepatocyte lineage population. In addi-tion, these cells did not exhibit detoxification function typi-cal of a more mature hepatocyte lineage cell. Therefore,studies were initiated to determine whether maintenance andaugmentation of hepatocyte-like function could be inducedin a secondary culture configuration. Hepatocyte lineagemaintenance was initially assessed by examining the dynam-ics of cell growth following removal of cells from their pri-mary EB culture and replating into tissue culturepolystyrene. A 5 � 104 cells from Day-17 EB cultures werereplated into one well of a 6-well plate and evaluated ondays 4, 6, 8 and 10 days post-replating. Cell numberincreased rapidly and confluence was reached at Day 6.Therefore, cells were replated into tertiary culture at 5 � 104

cells per well and continued to proliferate for the next 4days (Figure 1A).

Next, experiments were designed to evaluate the mainte-nance of function seen in EB-generated hepatocyte lineagecells by assessing in situ intracellular ALB and CK18expression. Secondary and tertiary cultures were initiated asoutlined earlier, and ALB and CK18 expression were quali-tatively assessed on 4, 6, 8, and 10 days post-replating usingindirect immunofluorescence with either primary anti-ALB/CK18 antibody or an immunoglobulin control serum andsubsequently fluorescently labeled secondary antibody.

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Images were captured using digital microscopy to determinethe percent of ALB and CK18 expressing cells within thecultures. The Day-17 EB-generated cells were 80% ALBpositive (Figure 1B) and 60% CK18 positive (Figure 1C).As depicted in Figure 1B, ALB expression was maintainedfor 6 days at �40% in secondary culture, however, expres-sion was not maintained past 6 days in tertiary culture.CK18 expression was maintained at minimal expressionlevels for 4 days in secondary culture but was absent onsubsequent days (Figure 1C). In secondary polystyrene cul-ture, EB-derived cells proliferated rapidly but could not sus-

tain ALB expression. Therefore, we explored the additionof soluble factors on proliferation and maintenance offunction.

OSM and SNAP supplementation

To investigate the effect of soluble factors previouslyshown to regulate hepatic function, replated cells were sup-plemented with either OSM or SNAP (Ehashi et al., 2005;Iwai et al., 2002). Cell numbers in the OSM-supplementedcondition were similar to that of the unsupplemented culturesand they increased dramatically. However, cells exposed toSNAP were generally characterized by slower growth rates.Because of the rapid growth seen in the unsupplemented andOSM cultures, at Day 6, cells were replated into tertiary cul-ture at 5 � 104 cells per well and continued to proliferatefor the next 4 days (Figure 2A). ALB expression was main-tained in the OSM-supplemented cultures for up to 8 days insecondary culture. The cells supplemented with SNAP alsomaintained some ALB expression up to 8 days in tertiaryculture but at a lower level. There was no significant expres-sion following 10 days in secondary culture in any condition(Figure 2B). Urea secretion was greatest at Day 8 in theOSM-supplemented condition; however, some secretion wasdetected at all experimental time points. The Day-17 EB he-patocyte-like cells exhibited a urea secretion rate of 50 lg/106 cells/day (Figure 2C). A summary of the hepatocyte-likefunctions tested is summarized in Table 1. CK18 expressionas well as other hepatocyte functions such as ALB secretion,glycogen storage, and CYP450-mediated detoxification wasnot detected at any level in the OSM- or SNAP-supple-mented cultures. Although addition of soluble factors main-tained ALB expression for up to 8 days in the secondaryculture, most hepatocyte functions were not maintained atany significant level and others were totally absent.

Collagen sandwich culture

To determine whether we could further augment and/ormaintain the function of the hepatocyte-like cells isolatedfrom the Day-17 EB culture, collagen sandwich culture, asystem which has been well studied for maintenance ofmature hepatocyte function (Dunn et al., 1989), was utilizedalone (GEL) and in conjunction with OSM (GOSM) andSNAP (GSNAP) supplementation. Cells cultured in a sand-wich configuration were characterized by a slower rate ofproliferation when compared with polystyrene culture. Cellsin the GEL and GOSM conditions reached maximum growthat Day 6 and cells in GSNAP by Day 8. Because of the low-proliferation rates in sandwich culture, the cells did not reachabsolute confluence and no tertiary culture was employed(Figure 3). As depicted in Figure 4, ALB expression wasdetected in all conditions for 6 days in secondary culture;however, expression was maintained at 10 days post-replat-ing, only in the GSNAP and GOSM conditions (Figure 4A).Although, the combination of collagen gel cultures andSNAP on Day 10 of secondary cell culture yielded an eight-fold increase in ALB positive cell population (Figure 4B)relative to the control, the cell number was fivefold lower(Figure 3). Even though the proliferation rate of SNAP-treated cells was significantly lower, cellular function betterresembled that of an adult hepatocytes population whichdoes not proliferate in vitro. A similar effect was evident inthe expression of CK18 where some expression was detected

Figure 1. Nonsupplemented secondary culture characterization.

(A) Cell number was assessed by counting total cells dissoci-ated following trypsinization. Cells were plated into tertiaryculture at 5 � 104 cells per well at Day 6. (B) Time course ofthe percentage of cells expressing albumin in polystyrene sec-ondary culture. Each data point represents the percentage ofcells with an intensity reading above 0. The average of threeexperiments is presented. All values were statistically signifi-cant when compared with the ES control. (C) Time course ofthe percentage of cells expressing CK18 in polystyrene second-ary culture. Each data point represents the percentage of cellswith a normalized intensity reading above 0. The average ofthree experiments is presented. All values were statistically sig-nificant when compared with the ES control.

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in the nonsupplemented, non-GEL (NS) condition for 4 daysbut it was expressed only in the GSNAP and GOSM condi-tions after 10 days (Figure 5A). However, while �45% ofthose expressed CK18 in the GSNAP condition only �20%were positive in the GOSM condition (Figure 5B). TheGEL, GOSM, and GSNAP conditions stored glycogen 10days into secondary culture. The NS conditions did not sig-nificantly stain for glycogen (data not shown).

Urea and ALB secretion, vital liver functions, were usedto asses mature hepatocyte-specific differentiated function. Adynamic profile of ALB secretion was established usingELISA analysis. Although at 4 days in secondary culturethere was an initial induction of ALB secretion in both theGOSM and GSNAP conditions, rates were significantlyhigher in the GSNAP condition on subsequent days whencompared with all other conditions. In addition, secretionwas maintained at 60 gg/106 cells/day after 10 days in sec-ondary culture (Figure 6A). The Day-17 EB-derived cellsdid not secrete ALB (data not shown) and had a urea secre-tion rate of 50 lg/106 cells/day. In secondary culture, allconditions maintained some urea secretion. However, the25 lg/106 cells/day observed in the GSNAP condition wassignificantly higher than any other condition at Day 10(Figure 6B).

At the end of the culture period, cells cultured in all con-ditions were characterized by a variety of cell morphologies.In all NG cultures, cells displaying elongated nonparenchy-mal morphology were present. In all double gel conditions,cells were assembled in random densely packed groupingsand exhibited tightly packed morphologies. However, in theGSNAP condition, there was a second morphology that wascharacterized by more than 95% of cells in groups of roundor square cells in a nonconfluent, loosely connected environ-ment (Figure 7).

Cytochrome P450 detoxification

Based on urea, ALB, CK18, and morphological analysis,the GSNAP and GOSM conditions were superior to otherconditions in maintaining hepatocyte-specific functions, with

Figure 2. OSM and SNAP supplemented secondary culturecharacterization.

(A) Cell number was assessed by counting total cells dissoci-ated following trypsinization. Cells from the three conditionswere plated into tertiary culture at 5 � 104 cells per well atDay 6. (B) Time course of the percentage of cells expressingalbumin in polystyrene secondary culture. Each data point rep-resents the percentage of cells with an intensity reading above0. The average of three experiments is presented. All valueswere statistically significant when compared with the ES con-trol. Asterisk (*) indicates statistically significant differences (P\ 0.05) from other conditions on that day. (C) Time course ofurea secretion rates in the supplemented conditions. The aver-age of three experiments is presented. All values were statisti-cally significant when compared with the ES control. Asterisk(*) indicates statistically significant differences (P \ 0.05)from other conditions on that day.

Table 1. Function Summary for Supplemented Cultures

NS SNAP OSM

Intracellular ALB þ þ þCK18 þ � �Urea þ þ þALB secretion � � �CYP450 � � �Glycogen � � �(þ) represents at least one time point of significant expression or

secretion function in the three conditions; (�) represents functions whichwere absent on all experimental days.

Figure 3. Collagen sandwich secondary culture characterization.

Cell number was assessed by counting total cells dissociatedfollowing trypsinization. Cell number in all GEL conditionswas assessed by counting the total number of cells dissociatedfollowing collagenase digestion and trypsonization. Asterisk (*)indicates time point at which the NS cells were passed to 5 �104 cells per well.

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GSNAP being somewhat more effective than GOSM. Todetermine whether GSNAP or GOSM was more efficient atyielding a hepatocyte lineage population, detoxification, anessential function imperative in defining a usable replace-ment hepatocyte cell population, was assessed. CytochromeP450 enzymes play a key role in detoxifying xenobiotics and

were used in these studies to assess hepatocyte function.These studies monitored the expression and stabilization ofBROD and methoxyresorufin o-dealkylase following induc-tion with 3-methylcholanthrene for 48 h in Day-17 EB-derived cells and for 10 days in secondary GSNAP and

Figure 4. Intracellular albumin characterization.

(A) Fluorescence and bright field images of intracellular albu-min on Day 10 of replated collagen sandwich cultures supple-mented with SNAP, OSM, and polystyrene cultures withoutsupplementation. (B) Time course of the percentage of cellsexpressing albumin in polystyrene secondary culture. Each datapoint represents the percentage of cells with an intensity read-ing above 0. The average of three experiments is presented. Allvalues were statistically significant when compared with the EScontrol. Asterisk (*) indicates statistically significant differen-ces (P\ 0.05) from NS and GEL conditions on that day.

Figure 5. Intracellular CK18 characterization.

(A) Fluorescence and bright field images of intracellular CK18on Day 10 of replated collagen sandwich cultures supplementedwith SNAP, OSM, and polystyrene cultures without supple-mentation. (B) Time course of the percentage of cells express-ing CK18 in the sandwich culture conditions. Each data pointrepresents the percentage of cells with an intensity readingabove 0. The average of three experiments is presented. Allvalues were statistically significant when compared with the EScontrol. Asterisk (*) indicates statistically significant differen-ces (P\ 0.05) from all other conditions on that day.

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GOSM cultures. BROD and MROD activity can be deter-mined from the enzymatic conversion of resorufin. This ac-tivity detected via increasing concentration of resorufin wasonly apparent after 10 days in secondary GSNAP culture(Figure 8A). The rate of production was similar to that ofthe Hepa 1-6 control (Figure 8B). This activity was not seenin GOSM cultures.

Discussion

The development of implantable engineered liver tissueconstructs and ex vivo hepatocyte-based therapeutic devicesis limited by an inadequate hepatocyte cell source. Differen-tiated pluripotent ES cells have been used to alleviate thecell source limitation problem but their utility is contingentupon generating the large number of cells and sustainingfunction for extended periods of time. In this study, we usedpreviously identified hepatocyte lineage cells (Novik et al.,2006) to evaluate the effects of OSM, SNAP, and collagensandwich culture on maintenance and augmentation of differ-entiated function already observed after EB-mediated differ-entiation. These studies indicate that maintenance offunction, characterized by intracellular ALB and CK18expression as well as urea secretion, was maintained in the

presence of SNAP and OSM when used in conjunction withcollagen sandwich culture. In addition, while ALB secretionand Cyp450 detoxification were not seen in the starting pop-ulation, they were induced after 10 days in the GSNAPcultures.

Although others have reported long-term maintenance ofhepatocyte-like function from ES sources differentiated invivo (Teratani et al., 2005a,b) there have been no reports ofmaintaining EB-derived hepatocyte-like function after theprimary differentiation is complete. In fact, most studies donot explore the function after the initial differentiation proto-col. A significant problem associated with ex vivo adult he-patocyte culture is the rapid loss of differentiated functionand morphology (Koide et al., 1989; Nahmias et al., 2007).In our studies, we saw a similar effect in that intracellularALB and CK18 expression was significantly reduced whencultured under standard tissue culture conditions and passedseveral times into secondary and tertiary culture. Addition-ally, because of the heterogeneity of the population cells thatare more likely to proliferate may be outgrowing the hepato-cytes and masking their function. It is possible that a combi-nation of these factors is contributing to loss of hepatocytefunction observed in standard tissue culture conditions. Tobetter maintain adult hepatocyte function in vitro, investiga-tors have shown that when cultured in collagen sandwiches,hepatocytes maintain ALB secretion and cell morphology forup to 42 days (Dunn et al., 1991). Furthermore, it has beenshown that the collagen sandwich configuration can selectagainst other liver nonparenchymal cell types such as stellatecells in favor of hepatocytes, indicating a potential selectiv-ity for hepatocytes within a heterogeneous population (Dep-reter et al., 2000). In fact, cells in the NG-NS conditionsexhibit morphology similar to that of elongated nonparency-mal cells such as stellate cells. The collagen sandwich con-figuration also provides a simple means of culturing the ES-derived hepatocytes when compared with techniques thatrequire several costly growth factors and growth surfaces. Inthese studies there was a marked difference in cellular mor-phologies observed in the collagen sandwich culture; how-ever, collagen sandwich culture alone could not maintainALB expression past the 6 days in secondary culture andhad limited CK18 expression.

Interestingly, when SNAP or OSM was added to the sand-wich culture, results were significantly altered. SNAP hasbeen shown to upregulate mitochondrial and differentiatedfunction in hepatocyte-like cells derived from ES cells(Sharma and Yarmush, submitted), and OSM has been iden-tified as a key morphogen in the transition of fetal to adulthepatocytes (Kamiya et al., 2006) as well as the adult liversregeneration in response to injury (Znoyko et al., 2005).Many investigators have measured ALB expression bothusing immunocytochemistry and RT-PCR to help identifyhepatocyte lineage cells in a variety of differentiation proto-cols (Choi et al., 2002; Kumashiro et al., 2005a; Miyashitaet al., 2002). Recently, ES cells transfected with green fluo-rescent protein (GFP) reporter gene regulated by ALBenhancer/promoter have been used to identify and isolate he-patocyte lineage cells during differentiation (Heo et al.,2006; Soto-Gutierrez et al., 2006; Teratani et al., 2005b;Yamamoto et al., 2003). In these studies there have beenreports of up to 70% ALB positive cells. However, as othershave previously shown that ALB may also be expressed inthe visceral endoderm as well as fetal tissue, it alone cannotbe used to confidently identify hepatocyte lineage cells

Figure 6. Albumin and urea secretion rates in sandwichculture.

(A) Time course of ALB secretion rates in the sandwich cul-ture conditions. The average of three experiments is presented.All values were statistically significant when compared withthe ES control. Asterisk (*) indicates statistically significantdifferences (P \ 0.05) from all other conditions on that day.(B) Time course of urea secretion rates in the sandwich cultureconditions. The average of three experiments is presented. Allvalues were statistically significant when compared with the EScontrol. Asterisk (*) indicates statistically significant differen-ces (P\ 0.05) from all other conditions on that day.

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(Asahina et al., 2004). The combination of sandwich culturewith SNAP supplementation not only maintained ALBexpression at all time points but also resulted in over 80%ALB positive cells indicating a relatively homogeneous pop-ulation about 4 weeks after differentiation induction. In addi-tion to ALB expression, CK18 expression was maintained at45% up to 10 days in secondary culture. Although this islower than the 60% positive population seen immediatelyfollowing EB differentiation, the GSNAP culture conditionwas the only CK18 expressing condition. A similar trendwas seen with urea secretion where after 10 days in second-ary culture, urea secretion in the GSNAP condition was notas high as the Day-17 EB culture but was significantlygreater than any other culture condition.

In addition to maintaining ALB, CK18, and urea secretion,the GSNAP condition also induced ALB secretion not seenat any significant level in Day-17 EB culture. ALB secretionfrom ES-derived hepatocyte lineage cells has been reportedpreviously (Gouon-Evans et al., 2006; Maguire et al., 2007;Soto-Gutierrez et al., 2006; Teratani et al., 2005a; Tsutsuiet al., 2006), and in the current studies it is first detected atDay 4 at 120 gg/106 cells/day and decreases to about 60 gg/106 cells/ day. Although this is significantly lower than thelevels of secretion seen in the Hepa 1-6 control, it is signifi-cantly higher than any other experimental condition eval-uated here and similar to previously reported ES-derivedhepatocyte-like secretion level.

In addition to the secreting function, detoxification wasalso detected via CYP450 metabolism in the GSNAP

Figure 7. Cellular morphologies.

(A) �10 magnification, phase contrast image of cells in the GEL condition. Cells with similar morphologies were observed in all Gel conditions.(B) �20 magnification, phase contrast image of cells described in A. (C) �10 magnification, phase contrast image of GSNAP cells in a nonconflu-ent, loosely connected environment. (D) �20 magnification, phase contrast image of GSNAP cells were greater than 95% of cells are in groups ofround or square cells indicated by white arrows. (E) �20 magnification, phase contrast image of NG cells. Cells with similar morphologies wereobserved in all non-GEL conditions.

Figure 8. Cytochrome P450 detoxification.

All graphs represent cells that have been in secondary culturefor 10 days. Hepa 1-6 were used as a positive control. (A)BROD activity is measured every 5 min via metabolism ofmethoxyresorufin to resorufin. Increases in resorufin concentra-tion indicate activity. (B) Averaged rates of production ofMROD and BROD based on total cell number.

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condition. Xenobiotic metabolism has been well character-ized in primary hepatocyte systems (Behnia et al., 2000; Royet al., 2001), and although there have been reports of induc-tion of CYP450 mRNA in ES-derived hepatocyte-like cells,there have been few reports on detoxification, a functionwhich would be critical for use of these cells in a LAD(Asahina et al., 2004; Soto-Gutierrez et al., 2006; Tsutsuiet al., 2006). Here, we used 3-MC for 48 h to induceCYP450 activities and observed that both BROD andMROD detoxification was observed at a level similar to theHepa 1-6 mouse hepatocyte carcinoma cell line.

Interestingly, in these analyses, most reported functionswere seen 10 days in the GSNAP condition. This allows usto maintain and augment function of spontaneously derivedEB-mediated hepatocyte-like cells. A possible mechanismmay be that the collagen sandwich is selected for the hepato-cyte-lineage cells while SNAP is acting to drive the cells toa more mature phenotype. In addition to maintenance, wewere able to show an increase in cell number from 5 � 104

Day-17 cells to 1 � 106 cells 10 days into secondary culturewhile still maintaining 80% ALB expression. Future experi-ments may be done to determine how long these cells canretain function following termination of SNAP supplementa-tion. In addition, we may also explore the duration and timeof initial exposure to the SNAP, which could uncover meth-ods to further increase cell mass while sustaining function.The fact that Day-17 EB cells can proliferate in the GSNAPcondition, while maintaining their hepatocyte-like character-istics, brings an added value to generate the large mass ofcells required for use in a LAD. Nevertheless, our data indi-cate a combination of maintenance and augmentation of he-patocyte-specific functions in conjunction with an increase incell mass in the GSNAP condition for up to 4 weeks postdif-ferentiation induction.

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