T E C H N I C A L B R I E F

Metabolically Functional Hepatocyte-Like Cellsfrom Human Umbilical Cord Lining Epithelial Cells

Han Hui Cheong,1 Jeyakumar Masilamani,2

Chun Yong Eric Chan,1 Sui Yung Chan,1 and Toan Thang Phan2–4

1Department of Pharmacy, Faculty of Science; 3Department ofSurgery, Yong Loo Lin School of Medicine; 4Tissue EngineeringProgram, Life Sciences Institute, Center for Life Sciences; NationalUniversity of Singapore, Singapore.2CellResearch Corporation Pte. Ltd., Singapore.

ABSTRACTThe primary hepatocyte is the best benchmark for drug biotransfor-

mation studies. However, due to the severe shortage of primary he-

patocytes, there is a need for alternative reliable cell source. This study

aims to isolate multipotent epithelial cells from the umbilical cord,

differentiate these cells into hepatocyte-like cells (HLCs), and inves-

tigate the potential of using the differentiated cells for in vitro drug

metabolism model. Human umbilical cord lining epithelial cells

(UCLECs) were subjected to hepatic induction over a period of 28 days.

HepG2 and cryopreserved human hepatocytes were used as control.

Immunohistological staining was carried out for a-fetoprotein (AFP),

albumin, cytokeratin 18 (CK18), and 19 (CK19). Glycogen storage

ability was assessed through periodic acid–Schiff stain. Reverse

transcription polymerase chain reaction was performed to examine

gene expression of hepatic nuclear factor 4a (HNF4a) and cytochrome

P450 isozymes 1A2, 2C9, 2D6, and 3A4. Ultra-performance liquid

chromatography tandem mass spectrometry (UPLC/MS/MS) was uti-

lized to analyze functional metabolic ability of the HLCs, where

CYP3A4 was chosen as the study focus and testosterone as the drug

substrate. After 28 days of induction, the fibroblastic phenotype of

UCLECs changed to rotund polygonal shape resembling that of he-

patocytes. Protein expression of AFP and CK19 was negative, while

albumin and CK18 expression was upregulated. Gene expression of

HNF4a, CYP1A2, CYP2D6, and CYP3A4 was observed but not for

CYP2C9. After 4 h of incubation with testosterone, UPLC/MS/MS de-

tected 2a-, 6b-, 15b-, and 16b-hydroxytestosterone. UCLECs are able

to differentiate into HLCs that express liver-specific markers, and have

functional metabolic capabilities.

INTRODUCTION

The liver, the body’s main detoxifying system, is a vital

organ for sustaining life. After absorption from the intes-

tines, all food and medications are first presented to the

liver, hence making the organ most vulnerable to any

xenobiotic insult. For individuals with acute liver failure, end-stage

liver diseases, or biliary atresia, the only effective treatment is liver

transplantation. Unfortunately, the availability of suitable liver for

transplantation is scarce as the number of patients in need of the

organ outstrips the number of donors. Based on the Organ Pro-

curement and Transplant Network data as of January 12, 2011,

there were 16,857 patients on the wait list for liver, while there were

only 5,779 donors in year 2010.1 Although recent advances in

technology have made bioartificial liver devices a possible alter-

native, the devices are still at the clinical trial stage. The major

stumbling block to establish these devices commercially is the lack

of reliable and sustainable cell source.2,3 As drug-induced liver

injury is the most common cause of acute liver failure, it is vital to

understand drug pharmacology and toxicology to screen out

potential hazardous xenobiotics during drug discovery and devel-

opment. The ideal benchmark for in vitro metabolism studies is

primary hepatocytes. However, maintaining primary hepatocytes

with its compendium of metabolism enzymes functional over the

duration of metabolism studies is difficult.4,5 Further, primary

hepatocytes are inconsistent between lots and individuals, mani-

festing wide variations in enzyme levels. These variations result

from nonstandardized tissue-handling procedures, as well as indi-

vidual differences in sex, age, health status, life styles, and other

environmental factors. This and limited supply of primary hepa-

tocytes pose additional challenges to the conduct of drug bio-

transformation studies and other researches. Substantial amount of

research has been invested in the search for alternative cell source

and in vitro drug study models. Immortalized cell lines, liver slices,

cryopreserved hepatocytes, and microsomal systems have been

reviewed for their use in drug distribution, metabolism, and

elimination studies.4–7 Each system has its limitations, such as

inconsistent supply of hepatocytes with stable characteristics for

large-scale, high-throughput applications and low expression of

cytochrome P450 enzymes.

ABBREVIATIONS: AFP, a-fetoprotein; APCs, adult stem or progenitor cells; CK18, cytokeratin 18; CK19, cytokeratin 19; CYP, cytochrome; DAPI, 40 ,6-diamidino-2-

phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; ESCs, embryonic stem cells; FBS, fetal bovine serum; HLCs, hepatocyte-like cells;

HNF4a, hepatic nuclear factor 4a; NHeps, normal human hepatocytes; OHT, hydroxytestosterone; PAS, periodic acid–Schiff; RT-PCR, reverse transcription polymerase

chain reaction; SPE, solid phase extraction; UCLECs, umbilical cord lining epithelial cells; UPLC/MS/MS, ultra-performance liquid chromatography tandem mass

spectrometry.

130 ASSAY and Drug Development Technologies MARCH 2013 DOI: 10.1089/adt.2011.444

Recent years have seen increasing number of studies reporting

the potential use of undifferentiated and differentiated adult stem

or progenitor cells (APCs) for cellular therapy.8–10 APCs have an

advantage over the more versatile embryonic stem cells (ESCs) in

that it avoids ethic controversies. Hence, further development of

APCs from bench top to clinical applications would be generally

more acceptable. Mesenchymal and epithelial APCs have been

isolated in various niches, such as the bone marrow, adipose tis-

sues, limbal stroma of the eye, umbilical cord, and breast milk.11–15

Our group had previously illustrated that mesenchymal progenitor

cells derived from human umbilical cord lining can be induced to

differentiate into adipocyte-like cells and thus have the potential

to be used as in vitro model to study adipogenesis and obesity.16

Using the same isolation method, the aim of this current study is to

isolate multipotent umbilical cord lining epithelial cells (UCLECs)

from the umbilical cord lining, induce them to differentiate into

hepatocyte-like cells (HLCs), and hence investigate the potential of

these HLCs to serve as a novel in vitro drug biotransformation

study model.

MATERIALS AND METHODSMaterials

All chemical reagents were purchased from Sigma Aldrich unless

otherwise stated. Dulbecco’s modified Eagle’s medium (DMEM)

without phenol red, DMEM, Medium 171, antibiotic-antimycotic

solution, secondary antibodies chicken-antirabbit and chicken-

antimouse Alexa Fluor 488, the SuperScript� III reverse transcrip-

tase, and Ultrapure agarose powder were from Invitrogen�. Fetal

bovine serum (FBS) was from Hyclone, Thermo Scientific. Cryopre-

served normal human hepatocytes (NHeps), hepatocyte culture me-

dium with supplements and growth factors (HCM� Bulletkit�), and

hepatocyte maintenance medium (HMM�) were purchased from

Lonza. HepG2 cancer cell line was from ATCC. Primary antibodies

were as follows: a-fetoprotein (AFP) was from Neomarkers; cyto-

keratin 18 (CK18) and 19 (CK19) were from Abcam�. Albumin pri-

mary antibody and Liquid DAB + substrate chromogen system were

from Dako; R.T.U. Vectastain� Universal Quick kit, which contains

2.5% normal horse serum, pan-specific biotinylated antibody, and

streptavidin peroxidase preformed complex, was from Vector

Laboratories, Inc. Formaldehyde and b-mercaptoethanol were from

ICN Biomedical, Inc. The RNA extraction RNeasy� Mini kit and

RNase-Free DNase set were purchased from Qiagen�. Oligonucleotide

and 10· TAE buffer were from 1st Base, Singapore. All primers,

ReactionReady� Human GAPD internal normalizer, and Reaction-

Ready Hotstart ‘‘Sweet’’ polymerase chain reaction (PCR) Master mix

were purchased from SABiosciences�. Gel Red� was from Biotium,

Inc. Loading dye, 100-bp DNA ladder, and CellTiter-Glo� Lumines-

cent Cell viability assay were from Promega. 6b-hydroxytestosterone

(6b-OHT) drug reference standard was from Cerilliant, while 2a-

hydroxytestosterone (2a-OHT), 15b-hydroxytestosterone (15b-OHT),

and 16b-hydroxytestosterone (16b-OHT) were from Steraloids, Inc.

Oasis� HLB solid phase extraction (SPE) cartridges were purchased

from Waters.

Source of UCLECsHuman umbilical cords were collected by CellResearch Corpora-

tion, Singapore, with informed consent of the mothers after normal

deliveries of healthy, full-term babies. All cells were isolated by

personnel of CellResearch Corporation, Singapore, as previously

described.17 Briefly, the Wharton’s jelly and blood vessels were

separated from the umbilical cord amniotic membrane by dissection.

The isolated umbilical cord lining was cut into small pieces and ex-

planted onto tissue culture dishes. Primary UCLECs were expanded in

proprietary medium, PTTe1, made up of Medium 171 supplemented

with 2.5% v/v FBS, 50 mg/mL insulin-like growth factor-1, 50 mg/mL

platelet-derived growth factor-BB, 5 mg/mL transforming growth

factor-b1, and 5 mg/mL insulin. UCLECs from three different umbil-

ical cords were randomly selected from the specimen bank of Cell-

Research Corporation, Singapore.

HepatogenesisThe UCLECs were plated at a density of 5,000 cells/cm2 and cul-

tured at 37�C in an atmosphere of 95% air and 5% carbon dioxide.

Upon reaching confluency at passage 3 or 4, the PTTe1 medium was

removed and replaced with HCM. The HCM was prepared using the

growth factors and supplements in the HCM Bullet kit as supplied by

Lonza, consisting of hepatocyte basal medium, epidermal growth

factor, ascorbic acid, transferrin, hydrocortisone, insulin, bovine

serum albumin, and gentamicin/amphotericin-B. Differentiation was

carried out over a period of 28 days, where the UCLECs were cultured

in HCM for 14 days, followed by HMM for another 14 days. The HMM

was prepared in accordance to Lonza’s recommendations by addi-

tion of accompanying supplements—insulin, dexamethasone, and

gentamicin/amphotericin-B—into the hepatocyte maintenance me-

dium. The media were refreshed every 2 to 3 days. HepG2 and NHeps

were used as controls. HepG2 cancer cell line was cultured in

accordance to supplier’s recommendations in DMEM containing 10%

v/v FBS. NHeps were seeded onto collagen-coated plates in accor-

dance to supplier’s recommendations and maintained in HCM for

48 h to allow for cell recovery and attachment before assays were

carried out. Media were refreshed every day.

Histological StainingImmunohistochemical: AFP. Briefly, the cells were fixed in 4% v/v

paraformaldehyde for 30 min, blocked with 2.5% v/v normal horse

serum for 20min, followed by incubation with primary antibody for

90min, and pan-specific secondary antibody for 30min. Following

incubation with streptavidin horseradish peroxidase, DAB + substrate

chromogen system was used in accordance to manufacturer’s recom-

mendations. Cell staining was visualized under light microscopy.

Immunofluorescence: albumin, CK18, and CK19. Cells were fixed

with methanol at -20�C for 10 min and then blocked with 2.5% v/v

normal horse serum for 20 min, followed by incubation with primary

antibody for 90 min, Alexa Fluor 488 secondary antibody for 30 min,

and 40,6-diamidino-2-phenylindole (DAPI) for 10 min. Cell staining

was visualized under Carl Zeiss Apotome microscope.

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Periodic acid–Schiff stain. Briefly, the cells were fixed in 4% v/v

paraformaldehyde for 30 min at room temperature and then per-

meabilized with 0.1% v/v Triton-X for 10 min. Cells were oxidized in

periodic acid solution for 5 min and washed several times with milliQ

water. This is followed by treatment with Schiff’s reagent for 15 min

and then washed under running tap water for 5 min. Their nuclei were

counterstained with hematoxylin for 1 min and then blued with 1%

v/v ammonia solution.

Reverse Transcription–PCRTotal RNA was extracted using the RNeasy Mini kit in accordance

to manufacturer’s instruction. Prior to synthesis of first-strand

complementary DNA (cDNA), the RNA samples were treated with

DNase I to eliminate genomic DNA contamination. The generated

cDNA was subjected to 40 cycles of thermal cycling where dena-

turation is at 95�C for 15 s, annealing at primer specific temperature

for 1 min, and extension at 72�C for 30 s. The annealing temperature

for CYP3A4 and hepatic nuclear factor 4a (HNF4a) primers is 55�Cand 60�C for CYP1A2, CYP2C9, and CYP2D6 primers. The PCR end

products were electrophoresed on 2% w/v agarose gel containing Gel

Red in 1· TAE buffer and visualized under UV light. Reverse tran-

scription–PCR (RT-PCR) was performed in triplicates for each um-

bilical cord lining sample.

Drug Metabolism AssayAssay for functional activity of the major cytochrome P450

enzyme 3A4 was performed on day 28 of differentiation using

testosterone, the FDA-preferred drug substrate for CYP3A4 assays.

The HLCs, NHeps, and HepG2 were incubated in DMEM without

phenol red containing either vehicle blank or 25 mM of testosterone.

After 4 h, an aliquot of 200 mL of the medium was taken from each

well and added to 400 mL of acetonitrile containing carbamazepine as

internal standard. Each sample mixture was subjected to SPE using

the Oasis HLB SPE cartridges. Eluents were dried at 37�C under

nitrogen stream, and reconstituted to 50 mL. Finally, 2 mL of each

sample was injected into the ultra-performance liquid chromatog-

raphy tandem mass spectrometry (UPLC/MS/MS) system for detec-

tion of hydroxylated metabolites of testosterone.

UPLC/MS/MS Instrument and ConditionsThe chromatographic system is made up of Waters Acquity UPLC�

system with Acquity UPLC� BEH C18 column (1.7 mm particle size,

100 · 2.1 mm i.d.), operated at 45�C, at flow rate of 0.5 mL/min. The

gradient used is as indicated in Table 1. Mobile phase A was milliQ

water with 0.1% v/v formic acid, and mobile phase B was acetonitrile

with 0.1% v/v formic acid.

The AB Biosystem Qtrap 3200 LC/MS/MS triple quadrupole mass

spectrometer was used for detection of the metabolites, operating in

the multiple-reaction-monitoring positive ionization mode, using

mass transitions m/z 305/269 for 2a-OHT and 6b-OHT, m/z 305/

97.3 for 15b-OHT and 16b-OHT, and m/z 237/194.2 for carbama-

zepine. 6b-OHT was chosen for quantification of the metabolite con-

centration using the Analyst version 1.4.2 software. As cell numbers in

different in vitro systems varies, peak intensities quantified by Analyst

were normalized using CellTiter-Glo Luminescent Cell viability assay

for unbiased comparison of 6b-OHT concentration.

RESULTSMorphology Changes and AFP Stain

Over the period of 28 days of differentiation, the morphology of

UCLECs changed from elongated, fibroblastic cells to rotund,

polygonal HLCs (Fig. 1A–E). In addition, arrangement of the cells

changed from streaky alignment to random scattering. Im-

munohistochemical staining for AFP was positive for naive UCLECs

(Fig. 1G). However, after 28 days of hepatic differentiation, AFP was

undetectable by the staining (Fig. 1H).

Periodic Acid–Schiff StainPeriodic acid–Schiff (PAS) stain was nearly absent at day 7 of

differentiation (Fig. 1J). Starting from day 14 of differentiation, the

fuchsia pink stain can be seen, indicating glycogen storage ability of

the HLCs. Maintenance of HLCs in HMM till day 35 did not show

increase in accumulation of glycogen compared with day 28.

Albumin, CK18, and CK19 StainGradual increase in albumin and CK18 expression was observed

as the cells differentiated. Expression of CK19 was low during the

initial 2 weeks of differentiation, and was hardly or not expressed

at all by 28 days of differentiation. The nuclei were observed to

change from oval to kidney-bean shape as illustrated by the DAPI

stain (Fig. 2).

Reverse Transcription–PCRGene expression of the hepatic marker HNF4a and the four

cytochrome P450 enzymes in question was observed in NHeps as

expected (Fig. 3). The HLCs were observed to express HNF4a; how-

ever, it is a splice variant of the primer used in this study as indicated

by the different band size from NHeps and HepG2. Expression of

Table 1. Ultra-Performance Liquid ChromatographyMobile Phase Gradient Profile

Time (min) A% B%

0.00 78 22

2.40 30 70

2.41 5 95

2.99 5 95

3.00 78 22

4.00 78 22

A: milliQ water with 0.1% v/v formic acid; B: acetonitrile with 0.1% v/v

formic acid. Flow rate of mobile phase at 0.5 mL/min, and column temperature

at 45�C.

CHEONG ET AL.

132 ASSAY and Drug Development Technologies MARCH 2013

CYP1A2, CYP2D6, and CYP3A4 was observed in HLCs, but no

CYP2C9 was detected. HepG2 expressed HNF4a strongly, while

CYP1A2 and CYP2D6 gene expression was relatively weaker com-

pared with that of NHeps. CYP2C9 and CYP3A4 gene expression was

faint in HepG2.

Testosterone MetabolismThe HLCs were able to metabolize

testosterone to its hydroxylated metab-

olites, and the retention times of the

peaks were identified to be 2a-OHT at

1.92 min, 6b-OHT at 1.52 min, 15b-OHT

at 1.45 min, and 16b-OHT at 1.84 min

(Fig. 4A). Normalized mean concentra-

tions of 6b-OHT formed by HLCs, NHeps,

and HepG2 were calculated to be

0.062 – 0.034 pM, 0.211 – 0.070 pM, and

0.007 – 0.011 pM, respectively (Fig. 4D).

DISCUSSIONIn vitro differentiation of APCs into

HLCs is not new. However, the APCs are

usually obtained from bone marrow. In-

vasive surgery is required to harvest APCs

from bone marrow, which inevitably de-

ters many from making donation as it

poses some extent of surgical risk and in-

convenience to the donors. Further, qual-

ity and quantity of the APCs isolated from

the bone marrow is affected by the donor’s

age.18 Therefore, the supply of APCs for

large-scale research and clinical applica-

tions is restricted.

On the other hand, umbilical cords

are easily available with less ethical

concerns as it does not involve the

termination of life. After delivery of the

baby, the umbilical cord is usually

discarded as biowaste. Therefore, the

umbilical cord can be harvested into a

collector containing the required

transport medium until subsequent

harvesting of progenitor cells. Hence, it

would not be difficult to seek consent of

parents to donate their newborn’s um-

bilical cord as there will be no addi-

tional distress nor inconvenience to the

mother and baby. It has been previously

shown that UCLECs are easy to isolate

and maintain,17,19 compared with pri-

mary hepatocytes. Using the tissue ex-

plant method and identical PTTe1

proprietary medium mentioned in

our study, Reza and team characterized the UCLECs and found

them to express Mucin1, p63, and most, but not all, ESC markers.19

p63 is a critical initiator of epithelial stratification, as well as key

regulator of cell adhesion and survival in progenitor cells in

squamous epithelium. This alluded to the highly proliferative and

Fig. 1. Morphological changes of umbilical cord lining epithelial cells (UCLECs) during hepaticinduction. (A–E) Under normal light microscopy, it can be seen that UCLECs gradually changedfrom fibroblastic phenotype to rotund, polygonal-shaped hepatocyte-like cells (HLCs) at re-spective time points of days 0, 7, 14, 21, and 28. (F–H) a-fetoprotein (AFP) immunohisto-chemistry stain of HepG2, naive UCLECs, and day-28 HLCs, respectively. Absence of AFPexpression at day 28 signified maturity of HLCs and nonmalignancy. Pictures were taken underbright-field light microscopy. (I) Periodic acid–Schiff (PAS) stain of naive UCLECs. ( J) PAS stainof hepatic differentiation at day 7 and subsequent 7-day interval time points. Differentiationwas extended to 35 days to investigate sustained glycogen storage. Gradual increase in fuchsiapink stain illustrated glycogen storage ability of HLCs. Cells were cultured in 35-mm dish. Colorimages available online at www.liebertpub.com/adt

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significant clonogenic abilities of UCLECs. In addition, UCLECs

were reported to have immunoregulatory effect,20 thus minimizing

the need for immunosuppressive drugs and risk of graft-versus-

host disease. In contrast, ESCs tend to form teratoma and elicit

immune responses when transplanted. UCLECs are therefore more

amenable to tissue regeneration and

cellular therapy compared with ESCs.

We illustrated in this current study that

UCLECs differentiated into HLCs with

metabolic capabilities. Over a period of 28

days of hepatic induction, the UCLECs

gradually changed from elongated, fibro-

blastic morphology to rotund, polygonal

shape. In addition, the positive PAS

staining is evident that the differentiated

UCLECs have glycogen storage abilities,

like the hepatocytes.

AFP is expressed only in fetal liver cells

and tapers off by adulthood. Expression of

AFP in the adult liver is an indication of

hepatocarcinoma. For that reason, AFP

expression functions as a hepatic marker

to distinguish the liver development

stage.21 Our observation of strong AFP

immunohistochemical staining of naive

UCLECs is thus not unexpected, as the

UCLECs used were at an early passage of 3

to 4 since postnatal. As mitotic expansion

progressed and UCLECs migrated outward

away from the initial cell clusters, it is

possible that the AFP expression is grad-

ually lost in subsequent daughter clones as

indicated by the faint AFP stain in UCLECs

further away from the initial cell clusters.

Since AFP expression is expected to taper

off in adults, this weakening of expression

in our study could be a reflection of regular

cellular development in primary cells. The

complete disappearance of AFP expression

in day-28 HLCs is heartening because it

demonstrates that the HLCs acquired the

adult hepatocyte phenotype and were less

likely to be malignant.

It is known that serum albumin is pro-

duced in the liver and hence the gradual

increase in immunofluorescence stain for

albumin is another indication of mature

hepatic characteristics. A study by Mizuno

and Singer reported concentration and

subsequent transfer of serum albumin

from endoplasmic reticulum to the Golgi

apparatus of HepG2 cells, leading to a

much higher concentration of the secre-

tory protein in the Golgi apparatus at cell steady state.22 This is in line

with our observation that the positively stained cell cytoplasm with

small particles clustering at the circumferences of the HLCs at days 21

and 28 is probably due to transportation and accumulation of al-

bumin to the Golgi apparatus of the cells. The gradual appearance of

Fig. 2. Immunofluorescence stain of albumin and cytokeratin expression during differentia-tion. Upregulation of albumin and cytokeratin 18 (CK18) and together with downregulation ofcytokeratin 19 (CK19) indicated a shift away from cholangiocytes and commitment towardhepatocyte lineage. HepG2 was used as positive control for the antibodies. Color imagesavailable online at www.liebertpub.com/adt

CHEONG ET AL.

134 ASSAY and Drug Development Technologies MARCH 2013

hepatic marker CK18 and disappearance of biliary marker CK19

renders that the multipotent UCLECs are moving toward commit-

ment into unipotent hepatocyte lineage. These respective up- and

downregulation of hepatocyte and cholangiocyte markers resembled

in vivo hepatogenesis.21

The presence of metabolic enzymes is an ultimate determination of

terminal stage of liver organogenesis. In our analysis of day-28 HLCs

at the genetic level by RT-PCR, varying amounts of cytochrome P450

transcripts were expressed. Although gene expression of CYP1A2,

CYP2D6, and CYP3A4 was much lower than that of NHeps, while

that of CYP2C9 was undetectable, the HLCs were considerably at the

terminal stage of hepatic differentiation. In a review by Donato and

workers, comparative expression of P450 transcripts in HepG2

against primary human hepatocytes was reported to be a mere 0.01%

Fig. 3. Reverse transcription polymerase chain reaction of cyto-chrome P450 isozymes and hepatic nuclear factor 4a (HNF4a). (M)100-bp DNA ladder, (Lane 1) HepG2, (Lane 2) day-28 HLCs, and(Lane 3) normal human hepatocytes (NHeps). Multiple bands ofHNF4a observed in day-28 HLCs are due to splice variants.

Fig. 4. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) analysis of testosterone metabolism by threedifferent in vitro systems. (A–C) Representative extracted UPLC/MS/MS chromatograms of testosterone metabolites detected in cellculture medium of day-28 HLCs (A), NHeps (B), and HepG2 (C). Retention time of each monohydroxylated metabolite was identified againststandard references and found to be 15b-hydroxytestosterone (OHT) at 1.45 min, 6b-OHT at 1.52 min, 16b-OHT at 1.84 min, and 2a-OHT at1.92 min. (D) Normalized mean concentration of 6b-OHT formed. Chart values were plotted based on (mean – SD) pM, n ‡ 3. Normalizedmean concentration of 6b-OHT formed by HLCs, NHeps, and HepG2 was calculated to be 0.062 – 0.034 pM, 0.211 – 0.070 pM, and0.007 – 0.011 pM, respectively. Three of the five samples for HepG2 had undetectable chromatogram peak and was regarded as 0.0 pM.

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ª MARY ANN LIEBERT, INC. � VOL. 11 NO. 2 � MARCH 2013 ASSAY and Drug Development Technologies 135

for CYP2C9, 0.03% for CYP1A2 and CYP3A4, and 1.57% for

CYP2D6.23 Despite the low gene expression of metabolic enzymes,

HepG2 is still widely used in hepatic studies24–27 due to the shortage

of human hepatocytes and the lack of better alternative cell-based

system.

Another indication of differentiation progressing toward mature

hepatocytes is the expression of HNF4a gene by HLCs. HNF4a, ex-

isting in several isoforms, is an important regulator of hepatogenesis

as it activates a cascade of transcription factors that define the gene

expression profile of mature hepatocytes.28 All HNF4a isoforms are

capable of binding to the regulatory regions of the cytochrome P450

genes with different activating properties,4 resulting in varying

expression of cytochrome enzymes.29 As mentioned in the manu-

facturer’s product information insert of the HNF4a primers, the

primers can also generate amplicons from splice variants or alter-

native transcripts. Hence, the observed difference in HNF4a band

size of HLCs from HepG2 and NHeps is most likely due to alternative

splicing of the gene in HLCs. In addition, the suppressed CYP2C9

gene expression in HLCs could be a consequence of low expression of

the HNF4a isoform needed to activate CYP2C9.

Due to different translation rates, mRNA, and enzyme stabilities,

data from RT-PCR experiment may not always reflect the concen-

trations of catalytically active enzyme present in cells.30,31 To eval-

uate the functional metabolic abilities of the HLCs, assay was carried

out using UPLC coupled with the 3200 QTrap mass spectrometer that

is equipped with triple quadrupole/linear ion trap capabilities to

detect metabolites. The presence of CYP3A4 activity is essential as

CYP3A4 is one of the major contributors to metabolic activities in

liver. Hence, CYP3A4 activity was chosen for this further examina-

tion. Testosterone was the selected drug substrate due to its high

correlation for CYP3A4 activity32 and is also an U.S. FDA-preferred

chemical substrate for in vitro drug development experiment in-

volving CYP3A4. Our study showed similar results with a develop-

ment and partial validation report of using UPLC/MS/MS method for

determination of testosterone and its metabolites in cryopreserved

human hepatocytes.33 Wang’s group reported that the UPLC/MS/MS

is a fast, sensitive, and specific method for separating and identifying

testosterone and its chemically similar metabolites, and the metab-

olites were best separated using Acquity UPLC� C18 column.33 They

detected 16a-OHT in their samples, which was not detected in any of

our samples. The major testosterone metabolite that is expected to be

formed by CYP3A4 is 6b-OHT, which was successfully detected to be

formed by all three cell lines. In addition, we found 15b-OHT in all

three cell lines, and the 2a-OHT and 16b-OHT metabolites were de-

tected in HLCs but not in NHeps. 15b-OHT has been reported to be a

metabolite of CYP3A4 minor pathway, while the rest of the hy-

droxylated testosterone metabolites have been reported to be cata-

lyzed to different extent by other cytochrome enzyme isoforms

CYP2C9, CYP2C19, and CYP2B6.34–36 Specific drug substrates for

each cytochrome enzyme isoform must be used to conclusively de-

termine the functional activities of the enzymes, which is beyond the

scope of this present study. The slight difference in the detection of

metabolites between our study and that of Wang’s group is likely due

to the different source of cryopreserved human hepatocytes, and

hence resulted in variation of metabolism enzyme content. Further, it

was reported that even when well-defined freeze-thaw conditions

were adhered to, viability of hepatocytes and attachment efficiency

declined after cryopreservation.5,37 In addition, there is a marked

downregulation of most cytochrome P450 gene transcripts upon

plating of the primary hepatocytes relative to freshly isolated primary

hepatocytes. In our study, during cell plating, there was significant

amount of hepatocytes remaining in suspension after the 48-h re-

covery and adherence allowance. Hence, the absence of 2a-OHT and

16b-OHT in NHeps of our study could be due to decreased cell via-

bility and alterations in the CYP3A4 gene transcription during

freeze-thaw and cell plating procedures.

After normalization against cell number, our data showed that

NHeps are the best in vitro system for metabolism based on the much

higher 6b-OHT concentration detected; an expected observation that

is in line with the unanimous acceptance of NHeps as the gold

standard for in vitro drug studies model. The HLC metabolism of

testosterone to 6b-OHT was *29% that of NHeps. Although me-

tabolism abilities of the HLCs were not as competent as desired, it is

unquestionably a better performance than HepG2 of only *3% of

NHeps. In addition, it was noted that of the five HepG2 samples that

were analyzed for presence of 6b-OHT, the metabolite was not

detectable in three samples. As the cytochrome enzyme activity in

HepG2 is low and it is tumorigenic, further development of this cell

line for clinical use is regarded as unachievable at this point.

In recent years, it has been reported that a newly derived human

hepatoma cell line HepaRG expresses hepatocyte-like functions, in-

cluding major cytochrome enzymes involved in drug metabolism.38–40

It was found that when HepaRG was seeded at high density, the

cytochrome P450 transcript expression was higher than that of

HepG2. Culture media containing 2% dimethyl sulfoxide (DMSO)

were needed to induce HepaRG to a more hepatocyte-like state and

enhance expression of the metabolizing enzymes. Without DMSO,

the HepaRG transcript expression for CYP2C9, CYP2D6, and

CYP3A4 was still relatively low at 22.1%, 0.8%, and 6.8%, respec-

tively, when compared with freshly isolated hepatocytes arbitrarily

set at 100%.39 In studies by other research groups where HepaRG was

demonstrated to be a promising in vitro model for metabolism studies,

2% DMSO was also added into the culturing media to maximize dif-

ferentiation.40,41 LeCluyse et al. reported that there was significant

increase in CYP3A4 activity of human hepatocytes in the presence of

DMSO at concentrations > 0.1% v/v.42 On the other hand, Easterbrook

et al. showed that DMSO inhibits CYP2C9 and CYP2C19, as well as

CYP3A4, in a concentration-dependent manner.43 Considering the

need for inclusion of DMSO into HepaRG culturing media for differ-

entiation and the conflicting reports of DMSO effect on metabolism

enzymes, the use of HepaRG as drug metabolism predictive models

may potentially result in complicated under- or overestimated data.

Moreover, HepaRG is after all a cancer cell line, and thus the response

to xenobiotics would inevitably differ from that of normal HLC line.

It has been shown that sandwich-culture configuration of isolated

hepatocytes between extracellular matrix such as collagen resulted in

CHEONG ET AL.

136 ASSAY and Drug Development Technologies MARCH 2013

reestablishment of matured hepatocyte functions, including trans-

porter activities,44,45 which is more reflective of in vivo pharma-

codynamics. Our data showed that in the absence of exogenous

extracellular matrix, the CYP3A4 enzyme of HLCs possessed intrinsic

enzymatic activity *29% that of NHeps. This encouraging finding

signifies that future hepatic differentiation of the UCLECs in sand-

wich-culture configuration and coculture with other accessory liver

cells would potentially stimulate an increase in CYP3A4 activity of

HLCs to a level that could be adequate for scale-up and application in

high-throughput drug-screening platform.

The above data taken together illustrated that the multipotent

UCLECs had differentiated into functional HLCs. With further studies

for other phase I metabolism enzymes, phase II metabolism abilities,

and membrane transporters, there is optimistic prospect of devel-

oping the HLCs for use as an alternative cell source for in vitro drug

metabolism studies.

ACKNOWLEDGMENTSThis work was supported by the National University of Singapore

research scholarship awarded to H.H.C. and the research support from

the National University of Singapore to S.Y.C. The authors thank

CellResearch Corp. (Singapore) for the generous gift of cord lining

epithelial cells and the proprietary expansion medium, Dr. Haishu Lin

for the use of SPE apparatus, and Dr. Tarang Nema for his assistance

in the SPE procedures.

DISCLOSURE STATEMENTNo competing financial interests exist.

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Address correspondence to:

Sui Yung Chan, BSc, MBA, PhD(Pharm)

Department of Pharmacy

Faculty of Science

National University of Singapore

18 Science Dr. 4

Singapore 117543

Singapore

E-mail: phacsy@nus.edu.sg

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