stably transfected human embryonic stem cell clones express oct4-specific green fluorescent protein...

Original Article

Stem Cells 2005;23:124–133 www.StemCells.com

Stably Transfected Human Embryonic Stem Cell Clones Express

OCT4-Specific Green Fluorescent Protein and Maintain

Self-Renewal and Pluripotency

Lesley Gerrard, Debiao Zhao, A. John Clark, Wei Cui

Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian, United Kingdom

Key Words. Human embryonic stem cell • OCT-4 • EGFP • RNA interference • Neural differentiation

Abstract

Correspondence: Wei Cui, Ph.D., Roslin Institute, Roslin, Midlothian, EH25 9PS, U.K. Telephone: 131-527-4200; Fax: 131-440-0434; e-mail: [email protected] Received May 6, 2004; accepted for publication August 23, 2004. ©AlphaMed Press 1066–5099/2005/$12.00/0 doi:10.1634/stemcells.2004-0102

Human embryonic stem cells (hESCs) are derived from

the inner cell mass of preimplantation embryos; they can

be cultured indefinitely and differentiated into many

cell types in vitro. These cells therefore have the ability

to provide insights into human disease and provide a

potential unlimited supply of cells for cell-based therapy.

Little is known about the factors that are important for

maintaining undifferentiated hESCs in vitro, however.

As a tool to investigate these factors, transfected hES

clonal cell lines were generated; these lines are able to

express the enhanced green fluorescent protein (EGFP)

reporter gene under control of the OCT4 promoter. OCT4

is an important marker of the undifferentiated state and

a central regulator of pluripotency in ES cells. These

OCT4-EGFP clonal cell lines exhibit features similar to

parental hESCs, are pluripotent, and are able to pro-

duce all three embryonic germ layer cells. Expression

of OCT4-EGFP is colocalized with endogenous OCT4,

as well as the hESC surface antigens SSEA4 and Tra-

1-60. In addition, the expression is retained in culture

for an extensive period of time. Differentiation of these

cells toward the neural lineage and targeted knockdown

of endogenous OCT4 expression by RNA interference

downregulated the EGFP expression in these cell lines,

and this correlates closely with the reduction of endog-

enous OCT4 expression. Therefore, these cell lines pro-

vide an easy and noninvasive method to monitor expres-

sion of OCT4 in hESCs, and they will be invaluable for

studying not only OCT4 function in hESC self-renewal

and differentiation but also the factors required for

maintenance of undifferentiated hESCs in culture.

Stem Cells 2005;23:124–133

Introduction

Embryonic stem (ES) cells are derived from the inner cell

mass of preimplantation embryos and retain the developmen-

tal potency of embryonic founder cells, being able to differen-

tiate into cells and tissues of all three germ layers in vitro and

in vivo. Therefore, ES cells have important implications for

providing insights into basic developmental biology. So far,

most studies on ES cells have been carried out in mouse ES

cells, as they were the only ES cells available until recently,

when human ES cells (hESCs) were generated from human

blastocysts. The establishment of hESCs provides resources

not only for studying basic human developmental biology but

also for their potential clinical applications. Subsequently,

much attention has focused on directing hESC differentia-

tion along specific developmental lineages and improving the

conditions for maintaining these cells in culture.

Gerrard, Zhao, Clark et al. 125

Although both human and mouse ES cells were originally

isolated and maintained by coculture on a mitotically inacti-

vated mouse embryonic fibroblast (MEF) feeder layer, they

may require different signals from the feeder cells for retain-

ing their undifferentiated status. This is suggested by several

studies [1–3]. In cultured mouse ES (mES) cells, the essential

function of the feeder layer is the provision of the cytokine,

leukemia inhibitory factor (LIF), and mES cells can be propa-

gated and maintained in culture provided that the appropriate

amount of LIF is added [4]. Stat3 activation by LIF is required

to sustain self-renewal [5, 6]. This pathway is thought to func-

tion in combination with additional signals that include OCT4

[7], Nanog [8, 9] and bone morphogenetic protein (BMP) sig-

naling [10] to maintain mES cells in the undifferentiated state.

However, in hESCs, the components involved in self-renewal

are poorly defined. In the presence of serum, LIF is not suffi-

cient to support the self-renewal of hESCs, and, even growing

on MEF feeders, hESCs require the addition of basic fibroblast

growth factor (bFGF) [1, 3]. A feeder-free method has been

developed whereby hESCs can be maintained on matrigel-

coated plates in culture medium conditioned from mitotically

inactivated MEFs and supplemented with bFGF [3]. It remains

unclear, however, what factors in the conditioned medium act

to support the self-renewal of hESCs. The generation of hES

reporter cell lines that contain a specific marker for undifferen-

tiated hESCs would provide invaluable tools for investigating

the factors that control self-renewal and allow much needed

improvements to be made to current culture conditions.

OCT4 (also known as OCT3, OCT3/4) belongs to the

POU (Pit-Oct-Unc) family of transcription factors [11] and

has been widely used as an important marker of undifferenti-

ated ES cells [10, 12]. OCT4 expression is associated specifi-

cally with embryonic carcinoma cells, embryonic germ cells,

and ES cells [11, 13]. It is essential for the development of the

pluripotent inner cell mass (ICM) in mouse embryogenesis

since targeted disruption of OCT4 in mice resulted in embryos

devoid of an ICM [7]. Quantitative analysis of OCT4 in mES

cells revealed that higher levels of OCT4 induce differentia-

tion of ES cells toward endodermal and mesodermal lineages,

whereas lower levels result in trophectoderm differentiation,

defining OCT4 as a key regulator of stem cell pluripotency

and differentiation [14, 15]. OCT4 is expressed at high levels

in undifferentiated hESCs and is downregulated upon differ-

entiation [2], demonstrating that OCT4 in hESCs has a similar

role in maintaining pluripotency. Therefore, OCT4 is a good

candidate gene for directing reporter gene expression.

Enhanced green fluorescent protein (EGFP) is an auto-

fluorescent protein that provides the opportunity to moni-

tor expression in living cells and can be quantified by flow

cytometry, confocal microscopy, and fluorimetric assays. In

addition, EGFP-expressing cells in a heterogenous culture

can be enriched by fluorescence-activated cell sorter (FACS)

analysis. There are two principal strategies to generate OCT4

-EGFP reporter cell lines. One is to use homologous recombi-

nation to generate “knock-in” cell lines to introduce a select-

able marker and the EGFP reporter into the endogenous OCT4

locus [16]. The other approach is the transgenic method,

whereby a plasmid containing a selectable marker and the

EGFP reporter under control of the OCT4 promoter can be

introduced into hESCs to generate clonal cell lines. The latter

approach was used in the work described here since the OCT4

promoter fragment has been extensively characterized and

known to drive tissue-specific expression [17–22].

Here we show that stably transfected clonal human ES cell

lines with the OCT4 -EGFP plasmid exhibit high expression

of EGFP in the undifferentiated state, which is downregulated

during differentiation. The expression of EGFP correlates

well with endogenous OCT4 gene expression in addition to

hESC surface markers. The OCT4 -EGFP cell lines, like their

parental cell line, have similar developmental potential and

are able to generate cell types of all three germ layers.

Materials and Methods

Construction of phOCT4-EGFP

The human OCT4 promoter (hOCT4pr, from 67539 to

71490 in human DNA sequence with accession number

AP000509) was amplified by polymerase chain reaction

(PCR) with primers: hOCT4pr-F (5'−TT CCC ATG TCA

AGT AAG TGG GGT GG-3') and hOCT4pr-R (5'-CGA

GAA GGC AAA ATC TGA AGC CAG G-3') using human

genomic DNA (Promega G3041; Promega, Southampton,

U.K., http://www.promega.com) as a template. The frag-

ment was cloned into TOPO vectors (Invitrogen, Paisley,

U.K., http://www.invitrogen.com), and the fidelity of the

DNA sequence was confirmed by bi-directional sequenc-

ing. The correct hOCT4 promoter was subsequently cloned

into vector pEGFP1 (BD Biosciences, Oxford, U.K., http://

www.bdbiosciences.com) by insertion into HindIII and AglI

restriction enzyme sites upstream of EGFP.

Maintenance and Transfection of hES Cells

H1 cells [1] provided by Geron Corp. (Menlo Park, CA,

http://www.geron.com) were cultured in medium condi-

tioned by mitotically inactivated MEF conditioned medium

(MEF–CM) supplemented with 8 ng/ml bFGF on matrigel-

coated plates, as previously described [3]. Cells were rou-

tinely passaged at a 1:3 dilution by treatment with 200 U/ml

collagenase IV (Invitrogen).

H1 cells were split 1:3 by 0.5 mM EDTA treatment 24

hours before transfection and seeded onto a matrigel-coated

126 Human ES Cell Lines Express OCT4-GFP Reporter Gene

six-well tissue culture dish. ApaLI linearized phOCT4-EGFP

plasmid (10 µg) was tranfected into H1 cells using Fugene

6 transfection reagent (Roche, Indianapolis, http://www.

roche-applied-science.com) at a DNA : Fugene ratio of 1:1.5,

according to the manufacturer’s instructions. G418 selection

was applied 48 hours after transfection at 200 µg/ml; after 3

weeks in selection, the surviving colonies were picked indi-

vidually to a 24-well plate and expanded.

Embryoid Body Formation and Neural

Differentiation

Embryoid bodies (EBs) were generated as previously

described [23]. Briefly, confluent ES cells in a six-well

plate were treated with 200 U/ml collagenase IV; then,

small clumps of cells were cultured in suspension in EB

differentiation medium (knockout-Dulbecco’s modified

Eagle’s medium [KO-DMEM; Invitrogen], 20% fetal calf

serum, 2 mM L-glutamine, and 100 × nonessential amino

acids). The culture was maintained in suspension for 5

days, and EBs were collected and plated onto 0.5% gela-

tin-coated chamber slides. The attached EBs were continu-

ously cultured in EB medium for another 4 days prior to

fixation for immunocytochemistry. For neural differentia-

tion, confluent ES cells were treated similarly as making

EBs, but they were cultured in N2/B27 medium [10]. The

cell aggregates were grown in suspension for 28 days, then

trypsinized and plated onto poly-L-lysine and laminin–

coated coverslips in N2/B27 medium for a further 5 days

before fixation for staining.

Immunocytochemistry and Flow Cytometry

Cells were either stained directly after phosphate-buffered

saline (PBS) washing (for cell surface antigen) or fixed at

room temperature with 4% paraformaldehyde for 10 min-

utes (EB for 20 minutes) and permeated with 100% etha-

nol for 2 minutes after washing with PBS. Cells were then

incubated with 10% goat serum to block nonspecific bind-

ing, followed by primary antibody for 1 hour. Secondary

antibody was applied for 30 minutes, and cells were washed

with PBS before mounting with Mowiol (Calbiochem, San

Diego, http://www.calbiochem.com). Monoclonal antibod-

ies against OCT4 (1:250; Santa Cruz Biotechnology, Santa

Cruz, CA, http://www.scbt.com), SSEA4 (1:5; Developmen-

tal Studies Hybridoma Bank of Iowa University, Iowa City,

IA, http://www.uiowa.edu/~dshb), and Tra-1-60 (1:12; gifts

kindly provided by Prof. P. Andrews, University of Sheffield,

Sheffield, U.K.) were used as markers for undifferentiated

hESCs. The rabbit polyclonal antibody against GFP (Molec-

ular Probes, Invitrogen) was used at a dilution of 1:250. For

differentiation, the following antibodies were applied:

monoclonal antibodies against β-tubulin III (1:1000; Sigma-

Aldrich Company, Dorset, U.K., http://www.sigma-aldrich.

com), α-fetoprotein (1:500; Sigma), and muscle-specific actin

(1:50; Dako, Glostrup, Denmark, http://www.ump.com/dako.

html); polyclonal antibody against nestin (1:200; Chemicon

International, Temecula, CA, http://www.chemicon.com).

Secondary antibodies used were goat anti-mouse tetra-

methylrhodamine isothiocyanate (1:100; Sigma), goat anti-

rabbit fluorescein isothiocyanate (FITC), goat anti-mouse

FITC, and goat anti-rabbit Texas Red (all at 1:400; Jack-

son Laboratories, West Grove, PA, http://www.jacksoni-

mmuno.com). Immunofluorescence was visualized and

captured using Nikon Eclipse TC2000-U or Digital Pixel

image analysis system.

Cells were prepared similarly for FACS analyses. Ten to

twenty thousand cells were acquired for each sample using

a FACScan (BD Biosciences) and analyzed with CELL-

QUEST software.

Karyotyping and Telomere Length Assay

Cells were incubated with 1 mg/ml colcemid for 3 hours,

then trypsinized, lysed with hypotonic buffer, and fixed in

glacial : acetic acid (1:3). Metaphase spreads were stained

with Giemsa, using standard methods. For each cell line, 10

metaphase spreads were analyzed. Telomere lengths were

checked by telomere restriction fragment Southern blotting

described previously [24].

OCT4 Knockdown by RNA Interference

ES cells were passaged by EDTA treatment onto matrigel-

coated six-well tissue culture plates at low density. Cells

were transfected with double-stranded small interfering

RNA (siRNA) oligonucleotides specific for either human

or mouse OCT4 at both 24 and 48 hours after plating, as

described [25]. Cells were analyzed for GFP and OCT4

expression 24 hours after the second transfection.

RT-PCR Analysis

Total RNA was extracted using Qiagen RNAeasy kit accord-

ing to the manufacturer’s instructions (Qiagen, Valencia,

CA, http://www1.qiagen.com). First-strand cDNA was

synthesized as described previously [24], and 1/10 of the

cDNA reaction mix was subjected to PCR amplification

with DNA primers selective for genes OCT4 (forward, 5'-

CTTGCTGCAGAAGTGGGTGG-AGGAA-3'; reverse

5'-CTGCAGTGTGGGTTTCGGGCA-3'), GATA-6 (for-

ward, 5'-GCAATGCATGCGGTCTCTAC-3'; reverse 5'-

CTCTTGGTAGCAC-CAGCTCA-3'), EGFP (forward,

5'-GTAAACGGCCACAAGTTCAGC-3'; reverse 5'-GGC-

GGATCTTGAA-GTTCA-3'), and internal control ß-actin


(forward, 5'-GATCAACT-CACCGCCAACAGC-3';

reverse 5' CTCCTCCTCCAGCGACTCAATCT-3'). PCR

cycles consisted of an initial denaturation step at 94°C for

5 minutes, followed by 26 amplification cycles at 95°C for

30 seconds, 61°C for 30 seconds, and 72°C for 45 seconds. A

final step of 72°C for 5 minutes was included. DNA contam-

ination was excluded by PCR on the RNA template without

reverse transcription.

Results

The human OCT4 promoter fragment amplified from

human genomic DNA by PCR spans base pairs –3917 to

+55, relative to the transcriptional start site, and is highly

homologous to the well-characterized mouse OCT4 frag-

ment [22]. Studies of the mouse promoter revealed that the

elements important for OCT4 tissue-specific gene expres-

sion reside in this promoter region [17–22]. These include

the distal enhancer that drives expression in the ICM and

primordial germ cells, and the proximal enhancer that

activates OCT4 in the epiblast [17]. The fidelity of the

human OCT4 promoter PCR fragment was confirmed

by sequencing and was then inserted into the pEGFP-1

reporter construct upstream of EGFP. The resulting con-

struct, phOCT4-EGFP, was linearized and transfected

into the H1 hESC line. Transfection was performed using

Fugene 6 transfection reagent in two wells of a six-well tis-

sue culture plate and, following selection, nine resistant

colonies survived. All the colonies expressed EGFP and

were subsequently expanded to establish clonal cell lines.

Transgene integration was confirmed by PCR and South-

ern blotting. Single-copy integration was found in all but

one clonal cell line (T9, data not shown). Three of these

OCT4-EGFP clonal lines (T5, T7, and T8) were character-

ized more extensively.

Figure 1. Live-image and immunostaining of OCT4-EGFP clonal cells. (A, D): Live images of hES OCT4-EGFP clonal cell line

T5 showed that EGFP expresses specifically in the undifferentiated hES colony but not in the surrounding stromal cells. (A): GFP

image. (D): corresponding phase-contrast image. (B, E): Colocalization of (B) GFP and (E) cell surface glycoproteins Tra-1-60 in

undifferentiated hES OCT4-EGFP clones. (C, F): Colocalization of (C) GFP and (F) cell surface antigen SSEA4. Inserts in E and F

are high magnification to show cell surface staining specificity. (G–I): Antibodies staining against three germ-layer markers: (G):

α-fetaprotein (AFP), (H): muscle-specific actin, and (I): β-tubulin III, all in the differentiated OCT4-EGFP cells via embryoid

body formation. Abbreviations: EGFP, enhanced green fluorescent protein; hES, human embryonic stem.


The OCT4-EGFP clonal cell lines grew normally, as did

their parental H1 cells, in the MEF-CM, exhibiting typical

undifferentiated hES colonies surrounded by differentiated

stromal cells (Fig. 1D). EGFP expression in these cell lines

was associated specifically with colonies of undifferentiated

hESCs and was absent in the surrounding differentiated stro-

mal cells (Figs. 1A, 1D). Antibody staining against glycopro-

tein antigen Tra-1-60 (Figs. 1B, 1E) and glycolipid antigen

SSEA4 (Figs. 1C, 1F) showed that the OCT4-EGFP hESC

lines also expressed these surface markers specifically in the

undifferentiated hESC colonies that were in correspondence

with EGFP expression. FACS analyses were carried out in the

T5 cell line and showed that, on average (n = 5), 70% of the

cells expressed high levels of EGFP (Figure 2A) and almost

all GFP-positive cells exhibited high levels of SSEA4 and

Tra-1-60 expression, which are 99% and 97%, respectively

(Figure 2C and 2D). SSEA4 and TRA-1-60 expression pat-

terns in OCT4-EGFP cells are very similar to the H1 parental

cell line. OCT4-EGFP clones have been propagated in culture

for approximately 1 year (50 passages) and have maintained

EGFP expression, normal hESC morphology, and markers.

They also exhibited stable telomere lengths, which are similar

to the parental H1 cells (Fig. 3A). It has been shown that these

cell lines retained the ability to generate all three germ layers

following EB formation and differentiation (Figs. 1G–I) and

are karyotypically normal (Fig. 3B).

Together, these data indicate that clonal hESC lines

expressing OCT4-EGFP have been generated and propagated

successfully without affecting the developmental potential

Figure 2. FACS analyses of OCT4-EGFP hESCs. (A): FACS

analyses of H1 and T5. Of the T5 cells, 70% expressed high lev-

els of EGFP, and no GFP expression was found in H1 cells. (B):

FACS analysis of T5 cells after neural differentiation in N2B27

medium. Only 6% of the cells retained high levels of EGFP

expression after 4 weeks differentiation. (C, D): FACS analyses

of hESCs surface antigens, SSEA4 and Tra-1-60, expression in

T5 cells showed more than 99% and 97%, respectively, of the

GFP-positive cells expressing these antigens. Abbreviations:

EGFP, enhanced green fluorescent protein; FACS, fluores-

cence-activiated cell sorter; hESC, human embryonic stem cell.

Figure 3. Telomere lengths and karyotype of OCT4-EGFP cell

lines. (A): Telomere lengths of OCT4-EGFP cell lines are similar

to their H1 parental cell line and remain stable after 10 passages.

(B): OCT4-EGFP cell lines are karyotypically normal. Image of

metaphase preparation from OCT4-EGFP clone T8 at 10 months

of continuous passage. Karyotype 46, XY normal male. Abbre-

viation: EGFP, enhanced green fluorescent protein.


and viability of hESCs. In addition, expression of EGFP does

not appear to be toxic to these cells, and no silencing of the

integrated transgene has been observed after long-term cul-

ture (over a year).

Since the OCT4-EGFP transgene integrated into the

genome randomly, it may not necessarily reflect endog-

enous OCT4 gene expression and act as a good marker for

undifferentiated hESCs. To determine if EGFP expression

driven by the OCT4 promoter in these cell lines correlates

with endogenous OCT4 gene expression, we carried out a

series of co-immunostaining with both antibodies against

OCT4 protein and EGFP during differentiation of OCT4-

EGFP cell lines toward the neural lineage. The OCT4-EGFP

cells were cultured in N2/B27 medium as cellular aggre-

gates in suspension for 28 days. Initially, all cellular aggre-

gates expressed similar levels of EGFP. After 21 days, EGFP

expression differed between and within cell aggregates.

Some were EGFP-positive, some were EGFP-negative, and

some exhibited patchy EGFP expression. When aggregates

were dissociated and plated onto poly-L-lysine and lam-

inin–coated plates after 28 days differentiation, a mixture

of groups of EGFP positive and negative cells was observed.

FACS analysis showed approximately 70% of cells became

EGFP-negative, whereas only 6% cells remained EGFP-

positive and 24% exhibited reduced levels of EGFP (Fig.

2B). Immunostaining of these cells showed that the majority

of the cells were EGFP-negative but positive for β-tubulin III

and nestin, the neural lineage markers; those cells remaining

EGFP-positive were negative for β-tubulin III and nestin but

positive with OCT4 antibody staining (Fig. 4). These results

suggest that EGFP driven by the OCT4 promoter faithfully

represents expression of endogenous OCT4 in undifferenti-

ated ES cells and during their differentiation.

To further establish the specificity of EGFP expres-

sion driven by the OCT4 promoter, RNA interference

was employed to specifically knock down OCT4 mRNA

in these cells. It has been reported that the introduction

of short double-stranded RNA into mammalian cells can

Figure 4. GFP expression associated with endogenous OCT4 expression during neural differentiation. (A): Staining cells with GFP

(green) and β-tubulin III (red) antibodies showed that cells remaining positive for GFP were negative for β-tubulin III and that β-

tubulin III–positive cells were negative for GFP. (B): GFP (green) was shown to be colocalized with endogenous OCT4 (red). (C):

Nestin-positive cells (red) were EGFP negative. Abbreviation: EGFP, enhanced green fluorescent protein.


Figure 5. siRNA knockdown OCT4 expression induces embryonic stem cell differentiation and downregulates EGFP expression in the

OCT4-EGFP cells. Cells transfected with siRNA oligonucleotides specific for OCT4, whether (A, B, E,F) mouse (mOCT4) or (C,D,

G,H) human (hOCT4), showed that hOCT4 siRNA dramatically decreased OCT4-GFP expression (D and G, green), which correlates

with the change in cell morphology (C) and the knockdown of OCT4 protein (G, yellow after overlay). Reverse transcriptase polymerase

chain reaction also showed reduction of OCT4 and GFP mRNA expression and induction of GATA6 expression in hOCT4 siRNA-

treated cells, as shown by the gel photo and histogram in (I). By contrast, the mOCT4 siRNA-treated cells retained EGFP expression (B,

E), as well as the OCT4 protein expression (E). A–D are live images; E–H are antibodies staining: GFP, green; OCT4, red; colocalization

GFP and OCT4, yellow; and DAPI, blue. Abbreviations: EGFP, enhanced green fluorescent protein; siRNA, small interfering RNA.


inhibit endogenous gene expression [26] and that transfec-

tion of hESCs with siRNA specific for human OCT4 results

in the downregulation of OCT4 expression and, hence, dif-

ferentiation of hESCs [25].

OCT4-EGFP cells were transfected with siRNA oligo-

nucleotides specific for human or mouse OCT4. It has pre-

viously been shown that the human OCT4 siRNA (hOCT4)

specifically targets the human OCT4 gene, whereas the

functional mouse OCT4 siRNA (mOCT4) exerts no effect

on human OCT4 expression [25]. To increase knockdown of

OCT4 in OCT4-EGFP cells, transfections were carried out

at both 24 and 48 hours after plating. A marked reduction

in EGFP expression was observed in those cells transfected

with the hOCT4 oligonucleotide 24 hours after the second

transfection (Figs. 5C, 5D, 5G, 5H), but not observed in

those transfected with mOCT4 (Figs. 5A, 5B, 5E, 5F). The

decrease in EGFP expression was also shown to correlate

with a change in cell morphology, whereby cells transfected

with hOCT4 (Fig. 5C) were found to have a more flattened

appearance than the mOCT4-transfected cells had (Fig. 5A).

Antibodies against EGFP and OCT4 showed that both EGFP

and OCT4 protein levels are reduced in hOCT4-transfected

cells (Figs. 5G, 5H) but not in mOCT4-transfected cells

(Figs. 5E, 5F) and that OCT4 and EGFP are generally colo-

calized. Some cells, however, retained EGFP proteins. One

explanation could be that reduction of OCT4-EGFP was not

a direct effect of OCT4 siRNA transfection but an indirect

result of OCT4 downregulation, which induced cell differ-

entiation. This reduction in protein levels was also likely due

to the extended half-life of EGFP, which is usually greater

than 24 hours. This was further supported by RT-PCR results,

examining the mRNA levels of OCT4 and EGFP. Both OCT4

and EGFP mRNAs were decreased at similar rates, about two-

fold lower in hOCT4 than in mOCT4 (Fig. 5I). In contrast to

the reduction of OCT4 and EGFP mRNA levels, GATA-6, a

marker associated with trophectoderm in early mouse devel-

opment and later mesoderm and endoderm development [27],

was clearly induced in hOCT4-transfected cells (Fig. 5I). Con-

trol RT-PCR reactions were performed in which no reverse

transcriptase was added to ensure true mRNA amplification

rather than contaminating genomic DNA (data not shown).

Together, these results confirm that EGFP expression

driven by the OCT4 promoter in OCT4-EGFP clonal hESC

lines truly reflects the endogenous OCT4 gene expression, and

that this correlates with the undifferentiated status of the cells.

Discussion

We have successfully generated OCT4 reporter human ES

cell lines by plasmid transfection and shown that these hES

clonal cell lines retain the normal hESC characteristics of

self-renewal and pluripotency. This correlates with the

results of early reports from other laboratories that clon-

ally derived hESC lines can be propagated for prolonged

periods in culture [28, 29]. Differentiating OCT4-EGFP

cell lines to neuronal cell types and specific targeting of

endogenous OCT4 downregulated EGFP expression further

confirming that the 4-kb OCT4 promoter contains appropri-

ate regulatory elements to drive developmentally specific

EGFP expression that correlates closely with endogenous

OCT4 gene expression. In addition, the OCT4-EGFP cell

lines retained features associated with normal undifferenti-

ated hESCs after long-term propagation, including normal

karyotype, immunophenotype, and the ability to generate

all three germ layers following differentiation.

Similar experiments have been reported to generate

hESC reporter cell lines for undifferentiation [16, 29]. Eiges

et al. [29] reported the generation of human reporter cell lines

in which EGFP expression was under the control of the Rex1

promoter. The Rex-EGFP was expressed in undifferenti-

ated hESCs and was downregulated during differentiation.

In this report, however, the expression of transgenic EGFP

and the endogenous Rex1 gene was not closely examined to

ensure that the transgene expression was truly representa-

tive of the endogenous gene, or whether transfected cell lines

were karyotypically normal and capable of long-term EGFP

expression. In another study, OCT4-EGFP reporter hESC

lines were created from a clonal H1.1 cell line, in which the

EGFP sequences were inserted into the 3' untranslated region

of the OCT4 gene by homologous recombination. The tar-

geted cells exhibited EGFP expression only in undifferenti-

ated ES colonies, and the expression was downregulated after

differentiation [16]. Similarly, this work did not address the

correlation between EGFP expression and endogenous OCT4

expression and other undifferentiated markers before and after

differentiation. In our studies, we generated OCT4-EGFP

reporter cell lines from parental H1 cells and characterized the

transfected clonal cell lines in more details, showing stable

EGFP expression in hES colonies after long-term culture and

that EGFP expression is representative of endogenous OCT4

gene expression. In our experiments, all colonies exhibited

EGFP expression after selection, though at various levels; no

transgene silencing was observed, presumably because the

selectable marker is linked to the EGFP transgene.

Since the first generation of human ES cells, much atten-

tion has been paid to improving the culture conditions for

hESCs, with particular emphasis on defining the factors that

are sufficient for maintaining hESC self-renewal and pluripo-

tency so that no animal feeder, matrix, or conditioned medium

is required [30–33]. It is known that OCT4 is important for

self-renewal, and its expression is tightly regulated in mES


cells [7, 14, 15]. In addition to OCT4, other factors have been

implicated in self-renewal of mES cells, including LIF/STAT3

[5, 6], nanog [8], and BMP4 signaling [10]. However, little

is known about which factors are important in hESCs self-

renewal. The OCT4-EGFP cell lines will be a powerful model

for such studies providing easy insight to the state of undiffer-

entiation and differentiation. This will enable other factors to

be examined in hESC lines and determine if they play a role in

hESC self-renewal.

OCT4 has also been reported recently to be important

for neurogenesis [34] and endoderm development [35]. The

OCT4-EGFP cell lines provide a marker that will allow the

OCT4 expression to be followed, quantified, and selected

in vitro. This marker will be an extremely valuable tool for

studying the function of OCT4 protein, not only in maintain-

ing hESC self-renewal but also during their differentiation. In

summary, the availability of these OCT4-EGFP cell lines will

provide important insight into improving the culture condi-

tions of hESCs and investigating the function of OCT4 in both

self-renewal and differentiation of hESCs.

Acknowledgments

We thank Prof. Peter Andrews, University of Sheffield,

for providing us with the Tra-1-60 antibody and Ms. Judy

Fletcher and Ms. Susan Craigmile for helping us with karyo-

typing as well as technical assistance with the FACS analy-

ses. We also thank the other members in the lab for their

support; this work could not be done without their help. The

work was sponsored by Biotechnology and Biological Sci-

ence Research Council and Geron Corporation.

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