evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse...

Toxicology in Vitro 22 (2008) 1273–1284

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Toxicology in Vitro

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Evaluation of in vitro cytotoxicity and paracellular permeability of intactmonolayers with mouse embryonic stem cells

Anthony R. Calabro, Roula Konsoula, Frank A. Barile *

St. John’s University College of Pharmacy and Allied Health Professions, Department of Pharmaceutical Sciences, Toxicology Division, 8000 UtopiaParkway, Queens, NY 11439, United States

a r t i c l e i n f o

Article history:Received 1 August 2007Accepted 28 February 2008Available online 29 March 2008

Keywords:In vitro cytotoxicityMouse embryonic stem cellsParacellular permeabilityTransepithelial electrical resistanceMTT assay

0887-2333/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.tiv.2008.02.023

Abbreviations: 3H-D, [3H]-D-mannitol; AC, acuteprimers in lower case), alpha fetoprotein; AR, amhydrocarbon receptor nuclear translocator; C-I, collagIV; ECM, extracellular matrix; EGF, epidermal growthgrowth factor receptor; FITC, fluorescein isothiocylaminin; GFs, growth factors (upper case); IC50, inhiinternal standard; KGF, keratinocyte growth factor; LILY, lucifer yellow; MA, multicellular aggregates; MEF,mES, mouse embryonic stem (cells); Oct-4 gene, Octfactor; PP, paracellular permeability; RC, Registry ofthelial electrical resistance or transmonolayer specifigene, transforming growth factor beta-receptor II; TJ,

* Corresponding author. Tel.: +1 718 990 2640; faxE-mail address: [email protected] (F.A. Barile).

a b s t r a c t

Mouse embryonic stem (mES) cells were induced to form intact monolayers in cell culture inserts, usingcombinations of extracellular matrix (ECM) components and growth factors (GFs). Progressive formationof intact monolayers was monitored using transepithelial electrical resistance (TEER) and passage ofparacellular permeability (PP) markers. The mES cells were initially inoculated on inactivated mouseembryonic fibroblasts (MEFs) plus leukemia inhibitory factor (LIF). At 75% confluence, cells were pas-saged in the absence of MEF and LIF to stimulate formation of rounded multicellular aggregates (MA).After 4 days, cultures containing MA were transferred to culture inserts coated with ECM componentsonly, and grown in the presence of selected individual GFs. An additional 10–14 days revealed confluentmonolayers with TEER values of 500–700 ohms cm2 (X cm2). Monolayers grown on inserts coated withECM components, such as fibronectin or collagen-IV, in the presence of epidermal growth factor or kerat-inocyte growth factor in the medium, yielded the highest TEER measurements when compared to cul-tures grown without GFs or ECM. Acute cytotoxicity (AC) studies with confluent monolayers of mEScells in 96-well plates indicated that there is a high correlation (R2 = 0.91) between cell viability and TEERfor 24-h exposure time. Also, decrease in TEER is inversely proportional with increase in PP of markers. Incomparison to standardized Registry of Cytotoxicity (RC) data and TEER measurements, MTT IC50 valuesfor mES cells are lower. Thus, at equivalent concentrations for the same chemicals, cell viability decreasesbefore the integrity of the monolayer is compromised. This system represents a novel approach for themanipulation of mES cells toward specific intact monolayers, as an in vitro model for biological mono-layer formation, and most importantly, for applications to cytotoxicity testing.

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1. Introduction

The Interagency Coordinating Committee on the Validation ofAlternative Methods (ICCVAM) and the National Toxicology Pro-gram (NTP) Interagency Center for the Evaluation of AlternativeToxicological Methods (NICEATM) recommended the development

ll rights reserved.

cytotoxicity; Afp gene (genephiregulin; Arnt gene, arylen type I; C-IV, collagen typefactor; Egfr gene, epidermal

anate; FN, fibronectin; LN,bitory concentration 50%; IS,F, leukemia inhibitory factor;mouse embryonic fibroblasts;-4/POU domain transcriptionCytotoxicity; TEER, transepi-c electrical resistance; Tgfbr2tight junction.

: +1 718 990 1877.

and submission of novel in vitro methods as simple predictivemodels for human acute cytotoxicity (AC). Optimization of thesesystems should also yield models that mimic gastrointestinalabsorption, dermal toxicity, and acute lethality in vivo, as well aspredict rodent LD50 values (ICCVAM publications 01-4499 and01-4500, 2001a,b). To address this problem, several investigatorshave recommended the ZEBET1 approach as a strategy to reducethe number of animals required for acute oral toxicity testing (Halleet al., 2000; Spielmann et al., 1999). The method uses in vitro cyto-toxicity data to determine the starting dose for in vivo testing, by cal-culating the standard regression between mean IC50 values andcorresponding acute oral LD50 data (Registry of Cytotoxicity, RC; ICC-VAM publication 01-4499, 2001a). The regression could then be usedto estimate the LD50 value of a new compound as the in vivo startingdose of a study. The application of this technique, however, is limitedby the lack of information on in vitro models for gastrointestinal, der-

1 Center for Documentation and Evaluation of Alternative Methods to AnimalExperiments, Germany.

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Table 1Comparison of IC50 data (mmol/l) for mES cells using the MTT assay and TEERmeasurements, with IC50 calculations (mmol/l) and LD50 values (mmol/kg) from theRC database for the same chemicals

Chemicals MTT 24-h TEER 24-h *RC IC50*RC LD50

Acrylamide 5.03 21 1.61 2.39Actinomycin 0.0009 0.08 0.000008 0.0057Antipyrine 13 130 11.6 9.56Cadmium chloride 0.1 0.14 0.0064 0.48Cupric sulfate 0.4 0.5 0.33 1.2Dimethyl formamide 69.9 700 114 38.3Doxorubicin 0.008 0.05 0.00033 1.2Glycerol 34.68 588 624.0 137Ibuprofen 0.145 0.1 0.52 4.89Lithium sulfate 9 97 34 10.8Manganese chloride 1.1 7 0.13 7.5Niacinamide 25.1 158 44 28.7Nickel chloride 0.277 1.0 0.27 0.81Propranolol 0.056 0.08 0.12 1.59Quinine HCl 0.084 0.11 0.075 1.72Salicylic acid 0.95 0.74 3.380 6.45Sodium dichromatea 0.075 0.048 0.00093 0.19Trichlorforon 0.96 2.08 0.27 1.75Verapamil HCl 0.09 0.44 0.10 0.22

*From the Registry of Cytotoxicity (RC) database (ICCVAM, 2001).a Dihydrate salt.

1274 A.R. Calabro et al. / Toxicology in Vitro 22 (2008) 1273–1284

mal and blood–brain barrier passage and biotransformation (Currenet al., 1998). In fact, monolayers of intestinal and colonic epithelialcells have been used as cell culture models for detecting transepithe-lial transport of drugs, paracellular permeability (PP) and otherintestinal responses to xenobiotics (Carriere et al., 2001). The effectof chemicals on the relationship between PP and AC testing in anin vitro model, however, is not well established.

We recently used Caco-2 monolayers (Konsoula and Barile,2005) and IEC-18 cells (Konsoula and Barile, 2007) as in vitro mod-els for comparing PP with AC of 20 chemicals. Caco-2, an immortalcell line originating from human colon, demonstrate transepitheli-al electrical resistance (TEER) values that are relatively higher thanIEC-18 monolayers cultured from the small intestine. The resultsfrom these studies revealed that finite IEC-18 cells grown on cellculture inserts were sensitive to toxic insult as immortal Caco-2cells, and cell viability decreased before membrane integrity wascompromised. IEC-18 cells, however, registered significantly lowerabsolute TEER values than Caco-2 cells. The lower resistance, aswell as the higher transport of chemicals in IEC-18 cells in culture,is analogous to the lower resistance of epithelial barriers withinthe small intestine (He et al., 1998). The diminished expressionof junctional proteins (ZO-1, occludin, e-cadherin) by IEC-18 sug-gests that junctional complexes are less tightly organized than inCaco-2 (Quaroni and Hochman, 1996). These features also explainthe higher TEER resistance of Caco-2 cells and prompt the need todevelop uniform, predictable in vitro models using diploid cells formeasuring AC and PP.

Mouse embryonic stem (mES) cells are derived from pluripotentcells of the early mammalian embryo and are capable of unlimited,undifferentiated proliferation in vitro (Evans and Kaufman, 1981).In chimeras with intact embryos, culturing of mES cells provide apowerful approach for introducing specific genetic changes intothe mouse cell line (Bradley et al., 1984). Pluripotency allows forthe ability of the cells to differentiate to the three embryonic germlayers. However, there are few reports demonstrating the differen-tiation of any ES cells to epidermal or epithelial structures. For in-stance, Yamada et al. (2002) describe the differentiation of mEScells into a functional gut-like organ in vitro that exhibits morpho-logical and physiological properties characteristic of the gastroin-testinal tract. Kuwahara et al. (2004) report that mES cellsundergo in vitro organogenesis by forming contracting gut-like or-gans from embryoid bodies (EBs). These structures are surroundedby epithelium, lamina propria, and muscularis. Ishikawa et al.(2004) further characterize interstitial cells of Cajal and smoothmuscle cells arising from EBs derived from mES cells. All of thesestudies were prompted by the developmental understanding thatin vivo epithelium undergoes continuous renewal by multipotentstem cells, which remain anchored to the crypt base. Thus a singleembryonic stem cell has potential to migrate and commit to all ofthe epithelial lineages (Kirkland and Henderson, 2001).

Several growth factors, particularly IL-3, (Wiles and Keller,1991), retinoic acid (Bain et al., 1995), and TGFb1 (Rohwedelet al., 1994), have been shown to direct linear specific differentia-tion of mouse stem cells. Epidermal growth factor (EGF), and therelated EGF family member, amphiregulin (AR), are mitogenicpolypeptides that induce differentiation into ectoderm and meso-derm (Gritti et al., 1999). Keratinocyte growth factor (KGF) is anepithelial cell-specific mitogen responsible for normal proliferationand differentiation of epithelial cells (Visco et al., 2004). In mostcases, the growth factors were applied to aggregates of ES cellsafter removal of leukemia inhibitory factor (LIF), a cytokine thatinhibits differentiation. In the absence of LIF, mES cells create intra-cellular contacts and initiate signaling and spontaneous differenti-ation (Furue et al., 2005). Keller et al. (2004) describe the inductionof epithelial- or epidermal-specific gene expression and differenti-ation using a combination of GFs plus extracellular matrix (ECM)

components in human ES cells. Based on these reports, we ex-plored the possibility that mES cells could be stimulated to differ-entiate and form confluent monolayers with high transmonolayerresistance when grown on porous inserts coated with ECM sub-strata. Moreover, the inclusion of mitogenic GFs in the media, inthe absence of MEF layers or LIF, could further promotedifferentiation.

Thus, two objectives directed our experimental plan. First, wesystematically designed a series of experiments aimed at deter-mining which combination of GFs and ECM coatings would guidemES cells toward the formation of intact cultured monolayers, asdetermined by TEER measurement and passage of PP markers. Sec-ond, we used the culture model to develop an in vitro system formeasurement of AC and PP. The contention is that the mES modelimproves the predictive ability of in vitro acute toxicity testing as-says for in vitro/in vivo correlations over Caco-2 cells because thelatter are a differentiated immortal cell line with higher TEER val-ues and greater resistance to chemical insults than mortal contin-uous cells. And, although IEC-18 cells are mortal diploid intestinalmouse cells, they do not form intact monolayers with sufficientTEER registry (150–200 X cm2). Finally, our mES cell model followson the successful development of prediction models using culturesof a pluripotent mouse embryonic stem cell line (embryonic stemcell test) for embryotoxicity testing (Genschow et al., 2000). Thus,the mES cells can be manipulated to form specific intact monolay-ers with critical tight junction (TJ) formation with significant TEERvalues, and can be applied to the development of AC testingmodels.

2. Materials and methods

2.1. Cell culture and chemicals

Cell culture liquid and powder media, and cell culture gradechemicals and supplements were obtained from Invitrogen Corp.(Carlsbad, CA, USA). Biocoat� cell culture inserts, Falcon� tissueculture multiwell plates, flasks, and other sterile disposable sup-plies were obtained from Becton Dickinson Labware (Bedford,MA, USA). All other test chemicals, including those listed in Table1, were obtained from Sigma–Aldrich (Sigma Life Sciences, St.Louis, MO, USA; Aldrich Chemical Co., Inc., Allentown, PA, USA).

Fig. 1. mES cells differentiate into rounded multicellular aggregates (MA) after r-emoval of LIF and in the presence of only ‘‘residual” MEF. The aggregates resembleEBs typically seen in suspension culture, are round and have a well defined border.

Fig. 2. Further culture in the absence of feeder layers and LIF induce MA to losetheir aggregate morphology, as cells migrate to the periphery with continuedproliferation.

A.R. Calabro et al. / Toxicology in Vitro 22 (2008) 1273–1284 1275

Chemicals used in the studies were suggested by the Registry ofCytotoxicity (RC; Halle, 2003); they were selected based on theverification of the data set (RC-II), and for their validity in estab-lishing a regression model between oral LD50 and IC50 values froma single mammalian cell line (ICCVAM publication 01-4500,2001b).

Mouse ES cells were propagated in culture with and withoutmitomycin-C treated mouse embryonic fibroblast layers [MEFs,3T3 Swiss mouse fibroblasts, CCL-92; American Type Culture Col-lection (ATCC), Rockville, MD, USA]. MEFs were seeded at104 cells/cm2 in T-75 culture flasks and grown in Dulbecco’s mod-ified Eagle’s medium supplemented with 10% newborn calf serum(DMEM-10), 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 4 mM glu-tamine, and 5% antibiotic/antimycotic solution, in an atmosphereof 5% CO2. When monolayers were 70% confluent, cells were trea-ted with 10 lg/ml mitomycin-C (mito-C) in DMEM-10 for 2 h at37 �C, after which the plates were washed and passaged with tryp-sin/EDTA. Pellets were replated at 1:3 dilution in T-75 flasks andallowed to attach overnight.

Mouse embryonic stem cells (mES cells, ES-D3, CRL-1934,ATCC) are derived from blastocysts of a 129S2/SvPas mouse. Allstudies were performed with cells at passage numbers 10–30.2

In our laboratory, mES cells were maintained in the undifferen-tiated state by frequent subculture (every 2 days) on confluentmito-C treated MEFs, and in DMEM containing 2 mM L-alanine-L-glutamine and 1000 U/ml leukemia inhibitory factor (LIF; recombi-nant human LIF, Millipore Corp., Temecula, CA, USA). The mediumwas adjusted to contain 3.5 g/l sodium bicarbonate, 4.5 g/l glucose,0.1 mM 2-mercaptoethanol, 0.5% non-essential amino acids(NEAA), and 15% fetal bovine serum (ES-DMEM). Medium changewas performed every day. Morphology of (a) mES cells in the pres-ence of feeder layers, and (b) multicellular aggregates (MA) anddifferentiated cells, in the absence of feeder layers, was docu-mented using phase contrast microscopy with the Leitz LabovertFS inverted phase contrast microscope equipped with the SPOT�

Insight 2.0 megapixel microscope camera, and processed withthe SPOT� advanced photographic software [although the forma-tion of MA in our cultures resembles embryoid bodies, the formerare rounded aggregates of cells that appear in monolayer cultures;thus the term EB is restricted to spherical aggregates that appearafter 4–5 days of growth in the absence of LIF or MEF in suspensionculture; see Figs. 1 and 2.

2.2. Assay procedures

2.2.1. Propagation of mES cells on culture insertsPrior to experiments, ES-D3 cells were maintained and propa-

gated in the undifferentiated state (Barile, 2007). To induce differ-entiation, LIF was removed from the media. Subconfluent mES cells(along with some residual inactivated MEFs) were passaged to cul-ture flasks without MEFs; MA formed after 4–5 days in the absenceof LIF or any additional MEF, and presented with multiple cell layerthickness over the few residual MEF cells (Fig. 1). Typically theaggregates are round and have a well defined border. Afterwards,the MA layers are trypsinized and transferred to flasks or cultureinserts where they lose their aggregate morphology and continueto proliferate (Fig. 2).

The culture containing MA, and residual mitotically inhibitedMEFs, were then trypsinized and seeded in 24-well plates fittedwith culture inserts (Isopore PCF polyester Millicell� culture plateinserts, 2.3 cm2 effective surface area, 105 cells per insert) with or

2 Although ATCC does not specify passage level, we routinely grow and maintaincultures under 30. This allows for shorter intervals between passages to maintainundifferentiated status; observable changes in culture characteristics are also lesslikely to occur.

without extracellular matrix (ECM) component coating (Biocoat�

inserts, Becton Dickinson Labware, Bedford, MA, USA). These com-ponents included: collagen type I (C-I), collagen type IV (C-IV),fibronectin (FN), and laminin (LN). In addition, cells were grownin the presence of one of the following human growth factors (Sig-ma Life Sciences, St. Louis, MO, USA): EGF, 400 ng/ml; AR, 200 ng/ml; transforming growth factor-b1 (TGFb1), 0.4 ng/ml; and, kerat-inocyte growth factor (KGF), 2 ng/ml. The differentiated MA cul-tures were grown for another 10–16 days, during which timedaily transmonolayer specific resistance (X cm2, see Section2.2.3) was measured using the Millicell-ERS� resistance system(Millipore Corp., Temecula, CA, USA) before and after incubationwith test chemicals.

2.2.2. MTT cell viability assayThe acute cytotoxic effects of 19 chemicals (see Table 1) on cell

viability were measured in confluent monolayers in 96-well plates,using the MTT assay (Dolbeare and Vanderlaan, 1994). This assaywas originally described by Mosmann (1983), and modified as pre-viously described (Schmidt et al., 2004; Konsoula and Barile, 2007).The tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT), is actively absorbed in a succinate-NADH+ mitochondrial-dependent reaction to yield a formazanproduct. The ability of the cells to reduce MTT provides an


indication of mitochondrial activity that is interpreted as a meta-bolic marker for cell viability.

Cultures containing MA were grown in 96-well plates until con-fluent, in ES-DMEM (components are noted above, Section 2.1) butin the absence of LIF, ECM, GFs, or any additional MEF. Confluentmonolayers were exposed to increasing concentrations of eachchemical (12-wells per concentration-group plus 1 control group;Table 1) for 24 h at 37 �C in an atmosphere of 7.5% CO2 in air. Inthe last hour of incubation, 10 ll MTT solution (5 mg/ml in DMEM)was added to each well. The medium was replaced with 100 lldimethylsulfoxide, agitated for 1 min at 25 �C, and the absorbancewas read at 550 nm on the BioTek FL600� fluorescence/absorbanceplate reader (BioTek Instruments Inc., Winooski, VT, USA). Cell via-bility is expressed as a percentage of the control group. Controlgroups consisted of cells in media (minus chemical) which wereprocessed identically and incubated simultaneously as treatedgroups. Parallel sets of wells without cells were incubated as pro-cess blanks.

For all assays, dosage range-finding experiments were per-formed. Every experiment was repeated at least thee times. Forexample, calculation of IC50 value for each chemical is based on aminimum of three experiments performed at separate times, eightgroups (concentrations) per chemical (including control andblank), and six separate measurements (samples) per group,(N = 3; sample number of measurements = 144). When the IC50

was not bracketed in the initial dosage range used for the chemical,the experiments were repeated and the concentrations were ad-justed as necessary. Values in figures are expressed as percent ofcontrol groups. (Additional protocol details involving chemicalsare noted below, Section 2.3).

2.2.3. Transepithelial electrical resistance (TEER) measurementsAs noted above, cultures containing MA, and residual mitoti-

cally inhibited MEFs, were trypsinized and seeded in 24-well platesfitted with Biocoat� culture inserts with or without ECM compo-nent coating. For TEER experiments, control inserts were notcoated and LIF was removed from all groups. In addition, the in-serts were divided into several groups and the cultures were al-lowed to proliferate and differentiate in the presence of ES-DMEM plus one of the following human GFs per group – EGF(400 ng/ml), AR (200 ng/ml), TGFb1 (0.4 ng/ml), or KGF (2 ng/ml)– for an additional 10–16 days. During this time daily transmono-layer3 specific resistance (X cm2) was measured. Results are detailedbelow and shown in Fig. 3. Experiments were repeated and the Nnumber generated as per the MTT assay (see Section 2.2.2 above).

2.2.4. Paracellular permeability (PP) studiesPP studies were performed as previously described (Konsoula

and Barile, 2005, 2007). Briefly, proliferating MA cultures wereseeded onto 12-well transwell polycarbonate inserts as describedabove. Cultures were incubated with the chemicals, which wereintroduced in ES-DMEM on the apical side of the insert (in contactwith the cell layer), for 24-h; low and higher molecular weight PPmarkers were introduced into the apical chamber in the last90 min of incubation. The markers included [3H]-D-mannitol([3H]-D; Mw = 182, 17 Ci/mmol, Perkin–Elmer, Boston, MA, USA),lucifer yellow (LY, Mw = 450, Sigma–Aldrich, St. Louis, MO, USA)and fluorescein isothiocyanate-dextran (FITC-dextran, Mw = 40–50 K, Sigma–Aldrich). Also, in order to stabilize and account forchanges in the osmolarity or pH of media from chemicals whoseTEER IC50 values exceeded 100 mmol/l, the incubating media wereadjusted with 100 ll,1 N NaOH and up to 25 lM HEPES buffer

3 The term transmonolayer is used here, as opposed to transepithelial, since thedifferentiation status of the MA is unknown. However, the TEER indication is widelyaccepted and understood.

(applicable for salicylic acid, ibuprofen and dimethyl formamide;Table 1).

For radioactive experiments, D-mannitol (0.1% w/v) was dis-solved in ES-DMEM, supplemented with [3H]-D (17 Ci/mmol),and added onto the apical side (0.5 ml) of the insert to a final con-centration of 1 mCi/l (Liu et al., 1999). ES-DMEM without [3 H]-Dwas added to the basolateral side. At the end of the exposure per-iod, an aliquot of basolateral medium was measured by liquid scin-tillation counting (Beckman LS5801 counter, Beckman Coulter Inc.,Fullerton, CA, USA). Blanks (inserts without cells) and controlgroups (minus chemical) were monitored simultaneously. Back-ground radioactivity was determined using DMEM, and dpm wascalculated based on the instrument’s counting efficiency (for[3H] � 45%).

LY and FITC-dextran fluorescent indicators were used at con-centrations of 1 mg/ml in ES-DMEM and applied to the apical sideof the insert, as per the radioactive experiments. At the end of theincubation and labeling period, an aliquot of the basolateral med-ium was collected and fluorescence intensity was measured withthe BioTek FL600� fluorescence/absorbance microplate reader.Experimental and process (blank) controls were monitored simul-taneously. The excitation and emission wavelengths for LY are430 nm and 540 nm, and for FITC-dextran, 487 nm and 518 nm,respectively. Relative cell permeability was expressed as a percentof untreated control groups. Experiments were repeated and the Nnumber generated as noted above (Section 2.2.2).

2.2.5. Gene detection for differentiation by RT-PCRTotal RNA was isolated from cell pellets using the Invitrogen

Micro-to-Midi total RNA isolation kit� (Invitrogen Corp., Carlsbad,CA, USA). Gene primers include: Oct-4/POU5f1 (POU domain tran-scription factor for undifferentiated cells and trophectoderm lay-ers), Afp (alpha fetoprotein for MA and EBs), Arnt (arylhydrocarbon receptor nuclear translocator for differentiated cells),Egfr (epidermal growth factor receptor for differentiated cells), andTgfbr2 (transforming growth factor, beta-receptor II for trans-formed cells). Forward and reverse primer sequences for all tran-scripts were purchased from SuperArray Bioscience Corp.(Frederick, MD, USA) (Ginis et al., 2004). RT-PCR was performedwith SuperScriptTM III one-step RT-PCR with platinum�Taq (Invit-rogen Corp., Carlsbad, CA, USA), using 0.4 lg RNA in each reaction(Kebache et al., 2002). PCR amplification was performed from amodification of the manufacturer’s protocol to allow for genequantification using 18 S ribosomal internal standard (IS; AmbionCorp., Austin, TX, USA). PCR products were amplified using a 1-min hot start at 94 �C, 1 cycle of 30 min at 50 �C and 35 cycles of15 s at 94 �C, 30 s at 55 �C, and 60 s at 72 �C, and a final extensionat 72 �C for 10 min. Expression products were separated using 2%agarose gel containing 0.5 lg/ml ethidium bromide for 60 min at100v. A 50 basepair DNA ladder (Invitrogen Corp.,) was used toestimate the size of the amplified bands. Band intensity was mea-sured using the FluoroChem 8000 Fluorescence Digital ImagingSystem� (FluoroChem Inc., Azusa, CA, USA). The bands werescanned for individual genes expressed in the PCR analysis andwere compared to the IS applied to the gel simultaneously and un-der identical conditions as the treatment sample. The informationwas then used to calculate the ratios in the presence of combina-tions of ECM and GFs.

2.3. Additional chemical solubility and protocol details

The 19 test chemicals were purchased from Sigma–Aldrich;they were stored desiccated at either 4 �C or �10 �C according tosupplier’s instructions. Soluble chemicals were dissolved in ES-DMEM stock solutions and concentrations were derived from ali-quots of the stock solutions. Solid chemicals that were insoluble

Fig. 3. Changes in TEER values for proliferating cultures of MAs in culture inserts in the presence of GFs (see insets) and four different ECM coatings (panels A–D). TEER valuesincreased early in culture with FN and collagen-coated inserts, whereas in the absence of ECM or in the presence of LN, this process is delayed by several days. Experimentswere repeated at least three times (n P 3).


in ES-DMEM, especially at the higher dosage levels, was improvedby the addition of a solvent to the incubating medium as follows(with pH adjusted to 7.4): salicylic acid and ibuprofen – 1 N NaOH;propranolol, verapamil, doxorubicin, actinomycin – 128 mmol/l(1% w/v) DMSO; quinine sulfate – 100 mmol/l (0.5% w/v) ethanol.In separate experiments we determined that none of the additivesinfluenced control group cytotoxicity or paracellular permeabilityof [3H]-D-mannitol. In addition, ethanol (100 mmol/l) served asthe negative control compound and is within +1 SE of the un-treated control groups (100% of control cell viability). Matchingconcentrations of solvents were also added to the correspondingtreatment groups where applicable.

2.4. Statistical analysis

The 50% inhibitory concentrations (IC50 values) were extrapo-lated from concentration–effect curves using linear regressionanalysis. The coefficient of determination (R2), regression analysis,slopes and the t-statistic (one- or two-tailed paired Students’ t-testwith the more stringent equal variances assumption) were alsocalculated for each set of data.

In addition, calculated regressions for the mES cells were com-pared with the RC regression (ICCVAM, 2001a,b) and with previouscytotoxicity data generated for Caco-2 and IEC-18 cells. The RCregression equation is based on the following formula:

logðLD50Þ ¼ 0:435� logðIC50Þ þ 0:625

where R2 = 0.67 for 347 chemicals in the RC database (Halle et al.,2000), and 0.97 for 11 of the 19 chemicals (ICCVAM, 2001a,b) usedin this study. If the regression line obtained with our model paral-lels the RC regression and is within ± log 5 interval, then the testis considered suitable to generate IC50 data to use with the RCregression for estimating starting doses.

For all experiments, every figure data point or IC50 value foreach chemical was calculated from a minimum of three experi-

ments performed at separate times, eight groups per chemical(each representing individual concentrations, including controland blank), six separate measurements (sample wells) per group(n = 3; sample number of measurements = 144). Because of thehigh sample repetitions, the SE for each data point presented inthe tables and figures are between 1% and 3%. In addition, propaga-tion of mES cells on culture inserts was performed bi-monthly overthe course of 2 years.

3. Results

3.1. Induction of formation of intact monolayers in culture inserts

Changes in TEER values for proliferating cultures of MA in cultureinserts in the presence of GFs (inset: amphiregulin, EGF = epidermalgrowth factor, KGF = keratinocyte growth factor, LIF = leukemiainhibitory factor, Tgfb2 = transforming growth factor beta-receptorII) and four different ECM coatings (panels: A = collagen type I,B = collagen type IV, C = fibronectin, D = laminin) are illustrated inFig. 3. TEER values increased early in culture with FN and collagen-coated inserts, whereas in the absence of ECM or in the presence ofLN, this process was delayed by several days. Also, highest TEER val-ues, up to 700 X cm2, were achieved with FN and C-IV coated inserts.In all cases, LIF and MEFs prevented formation of intact monolayerstructures, as evidenced by control baseline values.

TEER values of at least 500–700 X cm2 were achieved when MAwere grown in the culture inserts for 10–16 days without MEF orLIF. The development of high resistance intact monolayers wasaccelerated and best demonstrated when inserts were coated withC-IV or FN and when the culture medium was supplemented withKGF or EGF. The gradual increase and magnitude of the level ofTEER is an important finding and suggests that proliferating MA,in the absence of MEF or LIF, differentiate to form intact confluentselectively permeable monolayers (baseline measurements forcontrol cells were typically at 100–150 X cm2).


3.2. Cytotoxicity data

3.2.1. Comparison of PP data and TEER measurementsFigs. 4–6 illustrate results from PP experiments using PP trans-

port markers. The concentrations used are based on the IC50 datadetermined for the TEER experiments and range from .001 to five-fold of IC50 (Table 1). Each figure groups four representative chem-icals with corresponding transport of permeability markers atconcentrations based on the viability assays. The plots illustratethat paracellular transport of [3H]-D, as a PP marker, more closelyparallels the fall in TEER than LY or FITC-dextran. In every instance,transport of [3H]-D to the basolateral surface is proportional to thedecrease in transmonolayer specific resistance. PP data for the restof the chemicals are not shown but mimic the graphs in Figs. 4–6.

3.2.2. Cytotoxicity testing with confluent mES cells: comparison of cellviability and PP data

Table 1 compares the results generated with the in vitro as-says, and also contrasts them with standardized data from theRC database. The IC50 values were generated from the MTT as-say and TEER studies using confluent monolayers of mES cells.Cytotoxicity was determined using MTT cell viability assay in96-well plates; TEER was measured in confluent monolayersgrown on filter membranes, as described above. IC50 valuesfor each chemical were calculated from regression analyses ofthe plots of cell viability studies, the graphs of which areshown in Fig. 7. This graph illustrates concentration-effectcurves for 16 of the 19 chemicals using the 24-h MTT assay.The test statistic (t), for the calculation of each line of best

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fit, were significant for all plots as were the coefficients ofdetermination (R2 values greater than 0.90 for all chemicalsplotted, using log of concentration). Positive control chemical(actinomycin) represents the standard compound with the ex-pected most toxic effect (Table 1). As noted above, ethanoldid not show toxicity as the negative control chemical at100 mmol/l (0.5% w/v). We have also previously reported theIC50 for ethanol in a 24-h MTT assay using human lung cellsat greater than 500 mmol/l (Yang et al., 2002).

Table 2 summarizes the statistical analysis of the data in Ta-ble 1. The R2 and the slope (m) of the lines of best fit are indi-cated. R2 measures the degree of correlation between the sets ofdata, while the slope is an indication of the deviation of the plotof experimental values from a 1:1 (mM:mM) relationship. ANO-VA calculations revealed that significant differences exist amongthe data sets (F-significance <0.01); further statistical compari-sons revealed significant differences between groups where indi-cated (*Tables 2 and 3, one- or two-tailed paired Students’ t-test,P < 0.05).

The data reveal that MTT, as a measure of cell viability, cor-relates well with TEER measurements (R2 = 0.91). Cell viability,however, is a more sensitive indicator of cytotoxicity than TEER,as noted by the slope of the line (m = 0.08, significantly differentat P < 0.05, Table 2). This conclusion is based on the fact thatwhile R2 measures the degree of correlation between the setsof data, the slope is an estimate of the deviation of the plot ofexperimental values from a 1:1 relationship. Thus, in the calcu-lation of slope on a log scale, when m is less than 1.0, the lineis shifted to the right and the y-values are lower than corre-


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) verapamil, on PP in confluent monolayers of MAs. Concentrations are determined(range 0.001–fivefold, Table 1). Scale for % of control for PP markers (bars) is on leftat 100%. FITC = fluorescein isothiocyanate-dextran, LY = lucifer yellow, 3H = [3H]-D-

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Fig. 5. Effect of (a) actinomycin, (b) cadmium chloride, (c) cupric sulfate, and (d) doxorubicin, on PP in confluent monolayers of MAs. Concentrations are determinedempirically and based on IC50 measurements generated from MTT and TEER experiments (range .001–5-fold, Table 1). Scale for % of control for PP markers (bars) is on leftaxis; for TEER measurements (solid line), scale is on right axis. All control values are set at 100%. FITC = fluorescein isothiocyanate-dextran, LY = lucifer yellow, 3H = [3H]-D-mannitol, TEER = transmonolayer specific electrical resistance.


sponding x-values. This suggests that mitochondrial activity ismore likely to be altered before paracellular permeability iscompromised. Our model, for the combination of AC and PPmeasurements, thus allows for the differentiation between theconcentrations necessary for AC and those needed to interferewith the integrity of the intact monolayer structure.

3.2.3. Cytotoxicity testing with confluent mES cells: comparison ofcytotoxicity data with the in vitro IC50 data and in vivo LD50 RCdatabase

Similarly, we compared the IC50 values generated for 19chemicals using the MTT assay and TEER measurements, withIC50 values from the RC database (Table 1). The statistical analy-sis is tabulated in Table 2. Inspection of the data indicates thatthe MTT assay is more sensitive than the RC IC50 and LD50 val-ues, based on the slope of the lines (m = 0.07 and 0.35, respec-tively). Although these comparisons are not significantlydifferent, the slopes of the MTT values are consistently shiftedto the right of the RC IC50 and LD50 values, indicating lowernumbers (figures for Table 2 are not included but are similarto graphs of Fig. 8). The values also do not correlate well(R2 = 0.28 and 0.38, respectively). In addition, TEER values wereequivalent to the IC50 data from the RC database (m = 1.04),and less sensitive than the reported LD50s (m = 5.08). It is notsurprising, therefore, that TEER measurements were significantlydifferent from RC LD50s (Table 2; *P < 0.05; it should be notedthat the IC50 values from the RC database use the neutral red up-take assay as a cell viability marker and the 3T3 cell line).

3.3. Cytotoxicity testing with confluent mES cells: comparison ofcytotoxicity data with Caco-2 and IEC-18 in vitro IC50 data

Table 3 compares the IC50 MTT and TEER data generated withthe mES cells (from Table 1) with previously published data ob-tained from Caco-2 and IEC-18 cells using identical methodologies(Konsoula and Barile, 2005 and 2007, respectively). The statisticalanalyses are elucidated when the corresponding graphs are plotted(Fig. 8). High correlations (R2) are obtained when the same meth-odologies are compared between mES cells and the other cell lines.In particular, mES cells are significantly different (*) and more sen-sitive to the cell viability effects of the chemicals (m = 0.36) thanCaco-2 cells. Moreover, mES cells are more responsive to effectson cell viability when compared to IEC-18 cells (m = 0.26),although the paired comparisons of the IC50 data are not statisti-cally different. TEER IC50 measurements are almost identical be-tween the cell lines (Fig. 8c and d, and Table 3), suggesting thattoxic insult to intact monolayers of MA cultures are on the orderand magnitude of continuous human intestinal cell lines.

3.4. Gene expression as evidence of differentiation status of MAs usingRT-PCR

In order to gauge the state of differentiation of MA in culture in-serts, we used several gene transcripts that distinguish undifferen-tiated cells from their differential progeny. This also enabled us tocorrelate differentiation of MA cultures in inserts with the ability ofthe monolayers to produce high TEER resistance membranes.

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Fig. 6. Effect of (a) dimethylformamide, (b) glycerol, (c) manganese chloride, and (d) lithium sulfate, on PP in confluent monolayers of MAs. Concentrations are determinedempirically and based on IC50 measurements generated from MTT and TEER experiments (range .001–5-fold, Table 1). Scale for % of control for PP markers (bars) is on leftaxis; for TEER measurements (solid line), scale is on right axis. All control values are set at 100%. FITC = fluorescein isothiocyanate-dextran, LY = lucifer yellow, 3H = [3H]-D-mannitol, TEER = transmonolayer specific electrical resistance.


Differential expression of gene primers was quantified (Table 4)and compared to an 18S ribosomal internal standard (Figs. 9 and10). Fig. 9 shows representative expression of gene transcriptsfrom MA cultures grown on inserts for 14–16 days that werecoated with collagen type IV in the presence of KGF. Egfr expres-sion is highest among the genes amplified, compared to IS (lane3; ratio of Egfr/IS = 2.2, Table 4), while Tgfbr2 expression (lane 5,ratio = 0.8) and Afp (lane 1, ratio = 0.8) were lower (Egfr and Tgfbr2are expressed in differentiated cells and transformed cells, respec-tively, whereas Afp is expressed in transitional systems such asMA). Lowest expression was demonstrated for Oct-4, a markerfor undifferentiated mES cells (lane 4, ratio = 0.5). Consistent withthese results and as a biological control, MEFs showed the highestexpression of Tgfbr2 (5.2, Table 4), as a marker for immortal, trans-formed cells.

Fig. 10 illustrates KGF-induced expression from MA cultured onFN (lanes 1–5) and LN (lanes 7–11; lane 6 = 50 bp DNA ladder).Expression for Egfr was higher in the presence of FN (lane 3, ra-tio = 2.5) and LN (lane 9, ratio = 2.8) with lower expression of Oct-4 and Tgfbr2 (lanes 4 and 5, ratio = 1.1 and 1.0, respectively). EGF,however, in the presence of laminin, induces a heterogeneous pat-tern of gene expression since all of the ratio values are higher. Thissuggests that these cultures have not uniformly transformed fromthe MA stage. For instance, Egfr (lane 9, ratio = 5.7) and Oct-4 expres-sion (lane 10, 3.0), markers for differentiated and undifferentiatedcells respectively, demonstrated high band intensities with LN-coated inserts (Fig. 10 and Table 4). In FN coated inserts, calculationfor Afp/IS ratio (an MA marker, lane 1) is high (2.8).

4. Discussion

When ES cells are allowed to differentiate in suspension culture,they form spherical multicellular aggregates (EBs) that contain avariety of cell types (Yamada et al., 2002). Similarly, in the absenceof LIF or MEF after 4–5 days of growth, mES cells form roundedwell defined aggregates resembling EBs, containing multiple celllayers over the few remaining MEF cells (Fig. 1). As the MA coloniespropagate in the absence of feeder layers and LIF, they lose theiraggregate morphology and migrate peripherally (Fig. 2).

Limited evidence exists for the differentiation of mES cells intospecific populations, such as hematopoietic cells (Potocnik et al.,1997), cardiomyocytes (Klug et al., 1996), smooth muscle cells(Drab et al., 1997), and neurons (McDonald et al., 1999). Kelleret al. (2004) describe the induction of epithelial- or epidermal-spe-cific gene expression and differentiation using a combination ofGFs plus basal lamina substrata in human ES cells. Based on thesereports, we calculated that if mES cells are allowed to proliferateon porous inserts coated with ECM substrata in the absence ofMEF layers or LIF, we could direct the cells to differentiate andform confluent monolayers with high TEER resistance. In our firstattempts toward this end, we omitted MEF, LIF, and GFs with theintention of inducing spontaneous differentiation and formationof TJs based on their attachment to a basement membrane-coatedculture insert. Generation of monolayers with high TEER valueswas not extensive under these conditions (maximum TEER valuesreached 450 X cm2, Fig. 3, minus LIF), indicating minimal forma-tion of TJs. Thus, in the absence of GFs, the lack of production of

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Table 2Statistical comparison of MTT and TEER IC50 data from Table 1, and with IC50 datafrom the RC database for the 19 reference chemicals

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24-h MTT (mES) vs. 24-h TEER (mES) * 0.91 0.0824-h MTT (mES) vs. RC IC50 0.28 0.0724-h MTT (mES) vs. RC LD50 0.38 0.3524-h TEER (mES) vs. RC IC50 0.54 1.0424-h TEER (mES) vs. RC LD50

* 0.63 5.08

a Calculations based on values obtained from the RC database (ICCVAM, 2001).R2 = coefficient of determination for the regression analysis; m = slope of line of bestfit.* P < 0.05 (one- or two-tailed paired Students’ t-test with the more stringent equalvariances assumption).

Table 3Statistical comparison of MTT and TEER IC50 data from Table 1, and with IC50 datafrom Caco-2 and IEC-18 cells for the 19 reference chemicals

a

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24-h MTT (mES) vs. 24-h MTT (Caco-2)* 0.93 0.3624-h TEER (mES) vs. 24-h TEER (Caco-2) 0.93 0.8724-h MTT (mES) vs. 24-h MTT (IEC-18) 0.72 0.2624-h TEER (mES) vs. 24-h TEER (IEC-18) 0.92 0.75

a Comparisons based on cytotoxicity data for Caco-2 and IEC-18 cells previouslypublished (Konsoula and Barile, 2005, 2007, respectively). R2 = coefficient ofdetermination for the regression analysis; m = slope of line of best fit.* P < 0.05 (one- or two-tailed paired Students’ t-test with the more stringent equalvariances assumption).


higher resistance monolayers prompted us to incorporate GFs intothe protocol. These conditions generated intact monolayers withTEER values measured at 650–700 X cm2, suggesting intact conflu-ent monolayers with formation of TJs.

Results from RT-PCR experiments are in agreement with theTEER studies and indicate that MA cultures differentiate intospecific cell lineages (epidermal or epithelial cell lines) in 30–40% less time than expected in the absence of GFs and ECMcomponents. Highest expression of Egfr transcripts, as a markerof epidermal differentiation, also confirms the high TEER valuesobtained when culture inserts are supplemented with EGF orKGF (Fig. 3).

Further evidence for the influence of ECM and GFs on differ-entiation of MA in culture inserts is summarized as RT-PCR datain Table 4. The data reveal that FN and LN, in the presence ofKGF or EGF, induce higher expression of genes (Egfr and Arnt)corresponding to the differentiation of MA cultures with thehighest measured TEER values. In addition, Oct-4 expression (Ta-ble 4) is down-regulated for the corresponding combination ofGF/ECM, suggesting that by days 14–16, the MA cultures havedifferentiated, while Tgfbr2 expression is consistent with thepresence of transformed cells (‘‘MEF cells only” as biological con-trols). Thus semi-quantitative RT-PCR results suggest that differ-entiation of MA cultures in the presence of extracellular factorsis consistent with the gradual increase in TEER values duringthe culture period. Although there appears to be progressive dif-ferentiation of MA toward an epidermal cell lineage, expressionof Oct-4 and Afp in the same system suggest that the culturesare heterogeneous and correspond to various stages of differen-

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Table 4Ratios of gene expression to internal standard (IS) using RT-PCR of cultured MA in thepresence of extracellular matrix (ECM) and/or growth factor (GF) after 14–16 days onculture inserts

ECM GF Afp/IS Arnt/IS Egfr/IS Oct-4/IS Tgfbr2/IS

FN KGF 1.3 1.5 2.5 1.1 1.0EGF 2.8 1.3 1.8 0.9 1.8

LN KGF 1.2 1.5 2.8 2.9 1.3EGF 1.8 2.5 5.7 3.0 2.5

C-I KGF 0.8 1.2 2.3 0.9 0.9EGF 0.6 0.7 1.0 0.7 0.9

C-IV KGF 0.8 0.5 2.2 0.5 0.8EGF 0.9 0.8 1.1 0.7 0.8

MEF cells only – 2.0 3.0 3.9 0.7 5.2

Values represent the average of 2 or 3 experiments. Gene primers: Afp = alphafetoprotein, Arnt = aryl hydrocarbon receptor nuclear translocator, Egfr = epidermalgrowth factor receptor, Oct-4 = POU domain, class 5, transcription factor 1,Tgfbr2 = transforming growth factor, beta-receptor II; GF: EGF = epidermal growthfactor, KGF = keratinocyte growth factor; ECM: FN = fibronectin, LN = laminin, C-I/IV = collagen types I/IV, MEF = mouse embryonic fibroblast feeder layers.

Fig. 9. Expression of transcripts (lanes 1–5) for MA cultures grown on inserts co-ated with collagen IV (C-IV) in the presence of KGF; (lanes: 1. Afp = alpha fetopr-otein; 2. Arnt = aryl hydrocarbon receptor nuclear translocator; 3. Egfr = epidermalgrowth factor receptor; 4. Oct-4 = POU domain, class 5, transcription factor 1; 5.Tgfbr2 = transforming growth factor, beta-receptor II; 6. 50 bp DNA ladder). Ratio ofEgfr expression to internal standard (IS) was highest among the genes amplified(2.2), followed by Tgfbr2 (0.8), Afp (0.8), Arnt (0.5), and OCT-4 (0.5) (see also Table4, C-IV, KGF).


tiation. It is interesting to note that while the cultures appear toexpress markers of epidermal lineage, differentiation to an epi-thelial line has not been excluded.

The mechanism by which GFs and ECM influence cell differen-tiation is the subject of much interest. GFs are capable of inducinglinear specific differentiation. For instance, EGF is a mitogenic poly-

peptide that allows or induces differentiation into ectoderm(including skin) and mesoderm (Gritti et al., 1999), while KGF isa cell-specific mitogen responsible for normal proliferation and dif-ferentiation of epithelial cells (Visco et al., 2004). The TGFb familyof proteins, including the activins and the inhibins, bind to differ-ent cell surface receptors with varying affinities. They induce pro-liferation of mesenchymal and epithelial cell types whiledemonstrating anti-proliferative effects on endothelia, macro-phages and lymphocytes (Mercado-Pimentel and Runyan, 2007).Variants of the Tgfbr genes result in decreased TGFb-mediated

Fig. 10. Expression of transcripts for MA cultures grown on inserts coated with fibronectin (FN; lanes 1–5: 1. Afp, 2. Arnt, 3. Egfr, 4. Oct-4, 5. Tgfbr2) or laminin (lanes 7–11: 7.Afp, 8. Arnt, 9. Egfr, 10. Oct-4, 11. Tgfbr2; lane 6 = 50 bp DNA ladder), in the presence of KGF. Ratio of Egfr expression to internal standard (IS) was highest among the genesamplified, in the presence of FN (lane 3; Egfr/IS = 2.5) and laminin (lane 9; Egfr/IS = 2.8), with lower expression of Oct-4 in the presence of FN (lane 4; Oct-4/IS = 1.1) andlaminin (lane 10, Oct-4/IS = 1.9) (see also Table 4, FN, LN, KGF).


growth inhibition and an overall increase in cancer risk (Kaklamaniand Pasche, 2004). Amphiregulin (AR) is a member of the EGF fam-ily. AR is synthesized as a precursor; it is shed from the plasmamembrane by metalloproteases and is produced in a variety of tu-mors. Thus, AR is a pro-regenerative and survival growth factorthat displays a non-redundant role in cancer development, partic-ularly during acute liver injury (Berasain et al., 2007). In our exper-iments, therefore, it was reasonable to infer that enrichment of MAin culture with several specific GFs would provide an environmentfor cellular expansion, improved survival, or induced differentia-tion. Thus, our results suggest that GFs, particularly KGF in thepresence of C-IV substratum, induce differentiation of MA to formconfluent monolayers, with the subsequent formation of TJs. Thisaccounts for the high TEER values.

We obtained high regression values between MTT IC50 valuesand TEER measurements (Table 2), indicating that PP varies indi-rectly with cell viability – i.e. as cytotoxicity increases, TEER de-creases proportionately. According to the regression standards forthe RC database, cytotoxicity is predicted by our system. Althoughregression values are low, the slopes for all MTT assays vs. the RCIC50 values, RC LD50 values, and TEER measurements, are less than1.0, suggesting that cell viability measurements are more sensitivethan the latter. Consequently, computation of R2 alone is not suffi-cient for determining sensitivity of in vitro systems.

The results obtained from MA grown on cell culture inserts re-veal that they form intact monolayers with electrical resistancecomparable to that of Caco-2 and greater than that of IEC-18 cells.Differentiated cultures also demonstrate that there is a direct cor-relation between the disruption of the structural barrier and PP. In-creased transmonolayer passage of markers results whenmembrane integrity is compromised. Also, as with previous celllines, mES cells are as sensitive to toxic insult as immortal colonand finite small intestine cells. Moreover, all three cell lines revealsimilar TEER responses to chemical insult. Interestingly, analysis ofthese and previously published data demonstrates that mES andIEC-18 cells are permeable to all PP markers in contrast to Caco-2, the latter of which displays favorable passage of [3H]-D only(Konsoula and Barile, 2007). This may be explained by variableexpression of transporter/efflux systems present in Caco-2 cellswhich have been shown to alter paracellular and transcellular pas-sage of compounds, especially higher molecular weight chemicalssuch as FITC-dextran (Behrens and Kissel, 2003).

Paracellular transport of TJ markers, especially low-molecularweight [3H]-D, correlates precisely with decreases in TEER. SinceIC50 calculations for MTT experiments were generally lower thanTEER measurements, and PP of TJ markers increases systematicallywith a fall in TEER, it is reasonable to conclude that cellular viabil-ity is compromised before PP is affected. Thus, the mES/MA culture

insert model confirms our previous observations using immortalhuman colon cells and normal rat intestinal epithelial cells – i.e.,the system allows for the distinction between the concentrationsnecessary for AC and those needed to interfere with PP. It is alsoimportant to note that knowledge of a compound’s intestinal per-meability or absorption is not necessarily useful to classify cyto-toxicity [we have previously shown that comparison of 24-h MTTIC50 data for Caco-2 cells with available permeability indexes forthe 19 compounds tested reveals no correlation, suggesting thatpermeability indexes and cytotoxicity are not necessarily related(Ingels et al., 2004; Konsoula and Barile, 2005; Ruell et al., 2003).

Our findings for transformation of mES cells to MA, and subse-quent induction of differentiation to form intact monolayers withTJ formation, provides new possibilities for obtaining developmen-tally regulated monolayer structures for the replacement of epithe-lia lost to pathological complications. In addition, orientation ofundifferentiated stem cells toward lineages with distinct pro-grammed functions, presents the potential for understanding epi-thelial polarity, compartmentation, and barrier function. Mostimportantly, manipulation of ES cells toward specific lineages pre-sents a unique investigative opportunity for measuring the in vitroresponse to chemicals for toxicokinetic and cytotoxicity modeling.In particular, the construction of intact monolayers with TJ forma-tion may be of predictive value for epigenetic studies related toin vivo gastrointestinal development. It is in this specialized manip-ulation associated with stem cell differentiation wherein the advan-tage lies beyond other cell culture models, particularly since themodel more closely resembles the in vivo situation than Caco-2 orIEC-18 cells. Gene expression and immunohistochemical experi-ments are currently aimed at categorizing the stage of differentia-tion of cells in the presence of GFs and ECM, and their influence onTJ formation.

Acknowledgements

This work was supported in part by grants from the Interna-tional Foundation for Ethical Research (IFER, Chicago, IL, USA)and the Alternatives Research & Development Foundation (ARDF,Jenkintown, PA, USA). Parts of this study were presented at the an-nual meetings of the Society for In Vitro Biology, June 2006, and theU.S. Society of Toxicology, March 2007.

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