TERA2 and Its NTERA2 Subline:
Pluripotent Human Embryonal
I. ORIGINS OF NTERA2
TERA2 is one of the oldest extant cell lines established
from a human teratocarcinoma. It was derived
from a lung metastasis of a testicular germ cell tumour
and reported by Fogh and Tremp in 1975. However, its
pluripotent embryonal carcinoma (EC) properties
were not immediately obvious, in part because maintenance
of its undifferentiated EC phenotype requires
that cultures of these cells are passaged by scraping in
order to retain small clumps of cells instead of using
trypsinisation, which results in single cell suspensions.
In an early study of human teratocarcinoma-derived
cell lines we ourselves dismissed TERA2 as a potential
EC cell line (Andrews et al.
Initially, we failed to derive xenograft tumours from
TERA2 (Andrews et al.
, 1980). Nevertheless, after a
number of attempts we did succeed in obtaining a
single xenograft tumour after injecting a nude,
) mouse with TERA2 cells. Fortunately,
we explanted some of this tumour back into culture,
as well as fixing part for histology. To our surprise, histological
examination then revealed that the tumour
contained a variety of differentiated elements, as well
as embryonal carcinoma components; indeed it was a
teratocarcinoma. Meanwhile, it was evident that the
cells in culture, now named NTERA2 to designate their
passage through a nude mouse, did not resemble morphologically
the cells that predominated in the culture
of TERA2 that had been used to inoculate the host
mouse (Andrews et al.
, 1980). Indeed, the NTERA2
cells closely resembled other cells that we believed to represent human EC cells (Andrews et al.
From NTERA2 a number of cloned lines were
obtained by isolating single cells and culturing clones
from them. Detailed cytogenetic study and genetic
analyses using isozyme markers demonstrated clearly
that NTERA2 and its clones did indeed originate from
TERA2 (Andrews et al.
, 1984b). Subsequently, clones
were also derived directly from earlier passages of
TERA2 itself and these also exhibited EC cell properties
(Thompson et al.
, 1984; Andrews et al.
, 1985). It was
clear that the TERA2 line does contain EC cells but that
these can be lost by differentiation and overgrowth if
the cultures are maintained under suboptimal conditions.
Nevertheless, remaining subpopulations of EC
cells can be rescued from such cultures by cloning or
by growing of xenograft tumours in immunosuppressed
animals; evidently only undifferentiated EC
cells are tumourigenic.
Several single cell clones of NTERA2 were initially
studied, in particular NTERA2 clone B9, clone D3, and
clone D1. However, there was no obvious difference
among these and most subsequent studies have
utilised the clone NTERA2 cl. D1, which has also been
abbreviated NT2/D1 or sometimes simply NT2. The
different clones do exhibit slightly different karyotypes,
and it is certain that a small amount of genetic
drift occurs upon prolonged culture. Nevertheless, the
modal chromosome numbers of the NTERA2 clones,
and of TERA2 itself, are very similar, about 61 chromosomes,
including a variety of rearrangements.
Many of these rearrangements are common to all
clones, but new rearrangements continue to appear and characterise the individual clones (Andrews et al.
NTERA2 EC CELLS
The features of undifferentiated human EC cells are
best exemplified by another cell line, derived from a
testicular germ cell tumour, 2102Ep (Andrews et al.
1982). 2102Ep cells, and indeed a number of other
"nullipotent" human EC cell lines (Andrews et al.
1980; Andrews and Damjanov, 1994), tend to grow in
tightly packed colonies of small cells with little cytoplasm
and few prominent nucleoli within a nucleus
that comprises most of the cell. They exhibit doubling
times of about 20-24 h, and eventually form monolayers
from which domes may form and floating vesicles
bud off. The cells of these vesicles do not seem to be
significantly different from cells that remain attached
to the substrate. However, high-density cultures are
required to retain these featuresmtypically we seed
cultures at densities of at least 5 × 106
cells per 75-cm2
flask. At lower densities, the cells begin to flatten out
and evidently some differentiate into trophectoderms
(Andrews et al.
, 1982; Andrews, 1982; Damjanov and
NTERA2 EC cells behave similarly, although there
are differences. Maintenance at high cell densities is
essential but, in this case, for passaging, cultures
should be dispersed by scraping instead of using
trypsin and EDTAmcell:cell contact seems to be
required for the long-term retention of an undifferentiated
phenotype. Like 2102Ep, NTERA2 cells at low
density also flatten out and appear to differentiate, e.g.,
inducing expression of fibronectin, but they do not
seem to produce trophectoderm (Andrews, 1982;
Andrews et al.
, 1984b). Extraembryonic endoderm, evidenced
by the production of laminin, may be formed
(Andrews et al.
NTERA2 EC cells also share with other human EC,
and indeed embryonic stem (ES) cell lines, the expression
of characteristic marker genes, e.g., Oct4.
However, of particular utility is their expression of
surface antigens characteristic of undifferentiated
human EC cells (Andrews et al.
, 1983, 1984a,b,c, 1996)
). These include the glycolipid antigens stagespecific
embryonic antigen (SSEA) -3 and SSEA4, but
not SSEA1, the proteoglycan antigens TRA-1-60, TRA-
1-81, and GCTM-2, the liver/bone/kidney alkaline
phosphatase (L-ALP) associated antigens, TRA-2-49
and TRA-2-54, and also human Thy-1. This same
surface antigen phenotype is also expressed by humanES cells (Thomson et al.
, 1998; Reubinoff et al.
Draper et al.
, 2002), and several of the same antigens
(SSEA3, SSEA4, TRA-1-60, and TRA-1-81) are
expressed by the inner cell mass of human blastocysts
(Henderson et al.
In these respects, human EC cells differ from their
murine counterparts, which do not express SSEA3 or
SSEA4, or murine Thy-1 (Shevinsky et al.
Kannagi et al.
, 1983; Martin and Evans, 1974), but do
express SSEA1 (Solter and Knowles, 1978). Murine EC
cells do express high levels of alkaline phosphatase
(Bernstine et al.
, 1973), but that is not recognised by the
antibodies TRA-2-49 and TRA-2-54. The antibodies
TRA-1-60, TRA-1-81, and GCTM2 also do not appear
to recognise epitopes expressed by mouse cells. A
further difference between mouse and human EC cells
is the expression of class 1 major histocompatibility
complex (MHC) antigens, of which HLA is commonly
expressed by human EC cells (Andrews et al.
whereas H-2 is not expressed by mouse EC cells. In
fact, undifferentiated NTERA2 cells only express low
and variable levels of HLA-A,B,C, in contrast to other
human EC cells (Andrews et al.
, 1984b), but these levels
are increased markedly by exposure to interferon- 1
(Andrews et al.
III. DIFFERENTIATION OF NTERA2
NTERA2 EC cells differ from many other human EC
cells by their susceptibility to differentiation induced
by retinoic acid (Andrews, 1984) and to other inducing
agents such as hexamethylene bisacetamide (HMBA)
(Andrews et al.
, 1986, 1990) and the bone morphogenetic
proteins (Andrews et al.
, 1994). These agents
induce differentiation in distinct directions, although
the best studied is retinoic acid-induced differentiation,
which results in the formation of neurons as well
as other cell types (Andrews, 1984). By contrast, many
other human EC cells do not respond to retinoic acid
(Matthaei et al.
Within 24-48h after exposure to 10-5M
retinoic acid, NTERA2 cells commit to differentiate.
During the following 2 weeks, expression of the key
surface markers SSEA3, SSEA4, TRA-1-60, and TRA-1-
81 is eliminated in most cells, while they lose the characteristic
EC morphology, and begin expressing a
range of new markers. Prominent among these
induced markers are surface antigens SSEA1, A2B5,
and ME311 (Table I
), which appear to segregate to discrete
subsets of cells (Fenderson et al.
, 1987). At the
same time, expression of members of HOX gene clusters is induced (Mavilio et al.
, 1988). This induction of
HOX gene expression is dose dependent in a way that
relates to the position of the genes in the HOX cluster
(Simeone et al.
, 1990; Bottero et al.
, 1991). Thus, genes
located at the 5' end of the clusters tend to require
higher concentrations of retinoic acid for maximal
induction than genes located at the 3' end of the clusters.
This pattern appeared to relate to the expression
pattern of the HOX genes along the anterior-posterior
axis of the developing embryo and the postulated role
for retinoic acid in establishing that axis. However, differentiation
induced by HMBA, which causes a similar
elimination of the EC surface marker antigens, was not
accompanied by a comparable induction of the surface
antigens, or the HOX genes, induced by retinoic acid
(Andrews et al.
Differentiation of NTERA2 cells induced by retinoic
acid, and also HMBA, results in the appearance of susceptibility
of the cells to replication of both human
cytomegalovirus (HCMV) (G6ncz61 et al.
, 1984) and
human immunodeficiency virus (HIV) (Hirka et al.
1991). Neither virus is able to replicate in undifferentiated
cells, although they can gain entry. In the case of
HCMV, the block appears to lie directly in the inability
of the cells to support transcription from the major
immediate early promoter of the virus (LaFemina et al.
1986; Nelson et al.
, 1987). The nature of the block in the
case of HIV is less certain. However, both viruses
replicate readily in the differentiated derivatives of
NTERA2 cells, yielding fully infectious virions. In the
case of HIV, the entry of the virus into the cells does
not involve CD4 as the receptor (Hirka et al.
Among cells arising from the differentiation of
NTERA2, the neurons that appear after retinoic acid
induction are the most well defined and studied. These
appear only rarely after induction with HMBA or
BMPs (Andrews et al.
, 1986, 1990, 1994). A variety of
other cell types do appear in cultures induced with
each of these agents. These are poorly defined and are
not identified readily with specific cell types, although
they may include mesenchymal cell types, including
smooth muscle, fibroblasts, and chondrocytes (Duran et al.
Neurons were initially identified in retinoic acidinduced
cultures by their expression of neurofilaments
and toxin receptors (Andrews, 1984). Subsequently, a
detailed analysis of neurofilament expression confirmed
their neural identity (Lee and Andrews, 1986),
and electrophysiological studies indicated that they
expressed ion channels appropriate to embryonic
neurons (Rendt et al.
, 1989). The neurons formed by the
differentiation of NTERA2 cells can be purified readily
and have been widely used in a variety of studies of
human neural behaviour (Pleasure et al.
, 1992; Pleasure and Lee, 1993). Neural differentiation of NTERA2 cells
appears to follow a sequence of events that parallel
neural differentiation in the developing neural tube.
Thus an early marker induced following initial exposure
to retinoic acid is nestin, which is followed
sequentially by the expression of neuroD1, a HLH
transcription factor characteristic of postmitotic neuroblasts,
and finally by markers of mature neurons
such as synaptophysin (Pryzborski et al.
, 2000). The
appearance of the cell surface antigen A2B5 in differentiating
cells appears to be related to the neural
lineage and it seems that A2B5 expression is activated
shortly before cells leave the cell cycle. A2B5 is
expressed by the terminally differentiated NTERA2
neurons (Fenderson et al.
IV. REAGENTS, SOLUTIONS,
All cells are cultured in Dulbecco's modified Eagle's
medium (DMEM) (high glucose formulation), supplemented
with 10% fetal calf serum (FCS). DMEM may
be purchased from any reputable supplier of tissue
culture reagents. Samples of FCS should be obtained
from a number of suppliers and tested to identify a
batch that supports optimal growth of NTERA2 cells
and maintenance of an undifferentiated state, assayed
by the expression of appropriate markers (see later). A
batch of FCS that provides for good maintenance of
undifferentiated NTERS2 cells is not necessarily the
best for supporting differentiation. FCS should be
batch tested separately for both purposes.
Dulbecco's Phosphate-Buffered Saline (PBS)
For the following procedures, PBS without Ca2+
is used and may be purchased from any reputable
supplier of tissue culture reagents.
all-trans Retinoic acid can be purchased from Sigma-
Aldrich or from Eastman-Kodak. It should be stored in
the dark at -70°C, preferably under nitrogen. A stock
solution of 10-2M
retinoic acid (3mg/ml) in dimethyl
sulphoxide (DMSO) should be prepared and also stored
at-70°C. If prepared carefully under asceptic conditions,
this stock solution may be assumed to be sterile.
Hexamethylene Bisacetamide (HMBA)
A 0.3 M stock solution of HMBA should be prepared
in PBS and may be stored at 4°C. It may be sterilised
by passage through a 0.2-µm filter.
Purchase 3mm flint glass beads from a reputable
laboratory supplier. These should be washed in 10N
HCl and rinsed thoroughly with water. After drying,
about 20 should be placed in a Wasserman tube and
sterilised by autoclaving.
A solution of 0.25% trypsin in 1 mM
EDTA and PBS
free) may be purchased from any reputable
tissue culture supplier.
- Cytosine arabinoside: Prepare a fresh 1 mM stock
solution every 2 weeks and store at 4°C.
- Fluorodeoxyuridine: Store a 1 mM stock solution in
- Uridine: Store a 1 mM stock solution in water at
Matrigel is a proprietary product of Invitrogen and
consists of a mixture of extracellular matrix components
produced by an established tumour cell line. It
gels spontaneously at 37°C, but remains as a liquid at
4°C. It should be stored at-20°C on receipt. To use,
thaw a vial by standing on ice overnight. The thawed
Matrigel may be distributed into convenient aliquots
and refrozen once for further storage. To coat tissue
culture surfaces, the thawed Matrigel should be
diluted 1:30 in cold medium and enough to cover the
surface should be pipetted into the desired tissue
culture vessel (e.g., 1.5ml per 25-cm2
flask), which should then be incubated at 37°C for 2 h.
The Matrigel may then be aspirated and the flask is
ready for plating cells.
A. Maintenance of NTERA2 Stock Cultures
NTERA2 cells should be maintained at high cell
densities in DMEM plus 10% FCS at 37°C under a
humidified atmosphere of 10% CO2
in air. The FCS
should be batch tested to ensure that it is suitable for
supporting optimal growth of the cells (population
doubling times of approximately 20 h) in an undifferentiated
state, as assessed by morphology and expression
of surface markers (SSEA3, SSEA4, TRA-1-60, and
The cells should be harvested for subcultivation by
scraping, which can be achieved most easily with the use of 3-mm-diameter glass beads, stored in Wasserman
tubes. The contents of the tube may then be
tipped directly into a flask of cells to affect the detachment
of adherent cells as described.
B. Cryopreservation and Recovery of
- Choose a 75-cm2 flask of a well-grown, subconfluent
culture of NTERA2 cells (if harvested as a single
cell suspension, such a culture would yield about
20 × 106 cells).
- Aspirate some of the medium, leaving about 10ml
behind (alternatively, remove all the medium and
add 10 ml fresh medium).
- Tip about 20-30 sterile, acid-washed 3-mm glass
beads into the flask and roll them over the surface
of the flask. Rolling should be just vigorous enough
that the adherent cells detach in clumps.
- Gently triturate the detached cell clumps to reduce
their size to the order of 10-50 cells and transfer
approximately one-third of these to a new flask, to
which 20ml fresh medium should be added.
- The resulting culture should be maintained at 37°C under a humidified atmosphere of 10% CO2 in air;
it should be subcultivated as just described after
about 3-4 days.
NTERA2 cells may be frozen in a freezing mixture
consisting of 10% DMSO and 90% FCS.
C. Differentiation of NTERA2
- Harvest a well-grown, healthy culture of NTERA2
by scraping as described earlier.
- Centrifuge the cell suspension at 1000 rpm for 5 min.
- Remove the supernatant and resuspend the cells in
freezing mixture: use about 0.6ml for cells harvested
from a 75-cm2 flask.
- Distribute about 0.2ml of cell suspension in
freezing mixture to screw-cap cryovials and
transfer, in a cardboard box, to a -70°C freezer
- The next day, transfer the frozen vials of cells to a
liquid nitrogen freezer.
To Recover the Cells
- Thaw the cell suspension rapidly in a water bath at
- Dilute the cell suspension in 5 ml medium and centrifuge
at 1000 rpm for 5 min.
- Resuspend the cell pellet in fresh medium and
transfer to a new 75-cm2 flask; culture at 37°C as
NTERA2 may be induced to differentiate by a
variety of agents. However, the most widely used
agents are all-trans retinoic acid and HMBA. The following
protocol is used for the preparation of purified
neural cells from retinoic acid-induced cultures of
NTERA2 and is based upon the procedures of
Andrews (1984) and Pleasure et al.
D. Immunofluorescence and Flow Cytometry
- Harvest cells from a stock culture using
trypsin:EDTA and reseed at 1 × 106 cells per 75-cm2 tissue culture flask in 15 ml DMEM (high glucose formulation)
with 10% FCS and 10 -5M all-trans retinoic
acid diluted from the stock solution (10 -2 M) in DMSO.
[Note: 10-5M retinoic acid is a relatively high concentration;
lower concentrations may be used but certainly these have
different effects, for example, with respect to induction of
Hox genes (Simeone et al., 1990); also serum acts to buffer
the concentration of free retinoic acid, and lower concentrations
are necessary if used in conjunction with serum-free
- Maintain the cultures at 37°C and refeed with
fresh medium containing 10-5M retinoic acid every 7
days. [Note: Our standard protocol is to maintain the differentiating
cells continuously in retinoic acid. However, it
is possible to remove the retinoic acid after 2-3 days and
achieve full differentiation of the cells with the formation of
neurons, but there may be differences in the properties of the
- After 3 weeks harvest the cells with
trypsin:EDTA and reseed in a fresh flask at a split ratio
of 1:2 in DMEM with 10% FCS but no retinoic acid.
This is called "replate 1.'
- After 2-3 days add trypsin:EDTA (1 ml per 75-
cm2 flask) and observe under the microscope. As the
cells on the surface begin to detach (about I min), hit
the side of the flask sharply with the flat of your hand
to dislodge the loosely adhering, neural precursor
cells. Dilute the detached cells in fresh medium. [Note:
The object is to begin to purify the neural precursors from
the other differentiated derivatives, which adhere to the substrate
more strongly. Therefore, do not leave the
trypsin:EDTA on the cells long enough to cause all the cells
to detach, i.e., less than 3min.]
- Combine the harvested cells from several flasks
and reseed at 6-8 × 106 cells per 25-cm2 flask in DMEM
with 10% FCS and inhibitors (10-6M cytosine arabinoside,
10-5M fluorodeoxyuridine, 10-5M uridine).
This is called "replate 2.' Feed these cultures with fresh
medium including inhibitors every 3-4 days).
- After 1-2 weeks, treat the cultures with
trypsin:EDTA (1 ml per 25-cm2 flask). Monitor the cultures under a microscope and preferentially remove
the neurons that detach first (as in step 4). Dilute the
suspended detached neurons in 10ml fresh medium
and replate onto surfaces coated with Matrigel (see
later) at a density of about 105 cells/cm2, as required
for further experiments. The resulting neuronal cultures
(called "replate 3")can be maintained for several
weeks with refeeding every 4-7 days.
Monoclonal antibodies that are often used to assess
surface antigens on undifferentiated NTERA2 cells
and during their differentiation are shown in Table I.
These antibodies are generally available as culture
supernatants, purified antibodies, or ascites. They may
be used in a variety of immunoassays, but indirect
immunofluorescence, detected by flow cytometry, is
especially useful. The latter can be readily adapted for
sorting viable populations of cells for further functional
or developmental studies (e.g., Ackerman et al.
1994; Pryzborski et al.
, 2000). Whichever preparation
that is available should be pretitred on cells known to
express the relevant antigen. As a negative control, we
use the antibody from the original parent myeloma
of most hybridomas, namely P3X63Ag8 (Kohler and
Milstein, 1975). However, others may prefer a classmatched
nonreactive antibody if one is available or
else no first antibody at all.
Fluorescent-Activated Cell Sorting (FACS)
- Prepare a single cell suspension by harvesting
cells with trypsin:EDTA. After pelleting, resuspend the
cells in HEPES-buffered medium or wash buffer (PBS
plus 4% FCS and 0.1% sodium azide) at 2 × 106/ml.
- Choose antibodies and dilute as appropriate in
- Distribute the diluted antibodies at 50 µl per well
of a round-bottom 96-well plate.
- Add 50 µl of cell suspension (i.e. 105 cells) to each
50 µl of antibody.
- Seal the plate by covering with a sticky plastic
cover, ensuring that each well is sealed, and incubate
at 4°C, with gentle shaking, for 30-60rain.
- Spin the plate at 280g for 3 min using microtitre
plate carriers in a tissue culture centrifuge. Check that
the cells are pelleted and remove the plastic seal using
a sharp motion but holding the plate firmly to avoid
disturbing the cell pellet. Dump the supernatant by
inverting the plate with a rapid downward movement;
blot the surface and turn the plate over. Provided that
this is done in a single movement without hesitation,
the cells remain as pellets at the bottom of the wells. If
there are any concerns about pathogens contaminating a culture, supernatants can be removed by aspiration
rather than dumping.
- Wash the cells by adding 100µl wash buffer to
each well, seal, and agitate to resuspend the cells. Spin
down as described earlier. After removing the supernatant,
repeat with two further washes.
- After the third wash, remove the supernatant
and add 50µl fluorescent-tagged antibody, previously
titred and diluted in wash buffer to each well. FITCtagged
goat antimouse IgM or antimouse IgG, as
appropriate to the first antibody, may be used. Antimouse
IgM, but not antimouse IgG, usually works satisfactorily
with MC631 (a rat IgM). Affinity-purified
and/or F(ab)2 second antibodies may be used if
required to eliminate background.
- Seal the plate as described earlier and repeat the
incubation and washings as before.
- Resuspend the cells at about 5 × 105/ml in wash
buffer and analyze in the flow cytometer. The precise
final cell concentration will depend upon local operating
conditions and protocols.
- Harvest the hES cells using trypsin:EDTA as for
- Pellet the cells and resuspend in primary antibody,
diluted in medium without added azide, as determined by prior titration (100 µl per 107 cells). The
primary and secondary antibodies should be sterilized
using a 0.2-µm cellulose acetate filter.
- Incubate the cells with occasional shaking at 4°C for
- Wash the cells by adding 10ml medium and pellet
by centrifugation at 200g for 5 min; repeat this wash
step once more.
- Remove supernatant and flick gently to disperse the
pellet. Add 100 µl of diluted secondary antibody per
107 cells and incubate, with occasional shaking, at
4°C for 20min.
- Wash the cells two times as just described. After the
final wash, resuspend the cells in medium at 107 cells/ml. Sort cells using the flow cytometer according
to local protocols.
This work was supported in part by grants from the
Wellcome Trust, Yorkshire Cancer Research, and the
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