Propagation and maintenance of undifferentiated human embryonic stem cells.

Abstract

Human embryonic stem (hES) cells, like other stem cells, have the capacity to self-renew without differentiation. Although hES cells can be differentiated to many different tissue types in vitro, clinical uses have not yet been realized from the study of hES cells. Anticipation that these cells would be immediately useful for creating models of human disease has not yet been fulfilled. However, because of their self-renewing and pluripotential nature, hES cells indeed hold unique promise for many areas of research and medicine. A major problem complicating developments in hES cell research is the difficulty of propagating and maintaining these cells in vitro without differentiation. This review addresses this problem and potential solutions in detail. In addition, the current state of research regarding the growth and maintenance of hES cells is summarized, along with basic protocols utilized by our laboratory for the successful propagation, characterization, and investigation of hES cells.

Full text

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STEM CELLS AND DEVELOPMENT 13:243–253 (2004)
© Mary Ann Liebert, Inc.
Review
Propagation and Maintenance of Undifferentiated Human
Embryonic Stem Cells
MEGAN S. BODNAR, JUANITO J. MENESES, RYAN T. RODRIGUEZ, and MERI T. FIRPO
ABSTRACT
Human embryonic stem (hES) cells, like other stem cells, have the capacity to self-renew without
differentiation. Although hES cells can be differentiated to many different tissue types in vitro, clin-
ical uses have not yet been realized from the study of hES cells. Anticipation that these cells would
be immediately useful for creating models of human disease has not yet been fulfilled. However, be-
cause of their self-renewing and pluripotential nature, hES cells indeed hold unique promise for
many areas of research and medicine. A major problem complicating developments in hES cell re-
search is the difficulty of propagating and maintaining these cells in vitro without differentiation.
This review addresses this problem and potential solutions in detail. In addition, the current state
of research regarding the growth and maintenance of hES cells is summarized, along with basic
protocols utilized by our laboratory for the successful propagation, characterization, and investi-
gation of hES cells.
243
INTRODUCTION
H
ferentiation. In addition, hES cells are pluripotent and ca-
pable of differentiating to cells of all three germ layers
of the embryo (Fig. 1) (1–6). Because of their self-re-
newing and pluripotent nature, hES cells hold unique
promise for many areas of research and medicine (7–14).
Prior to the derivation of hES cells or animal in vivo
models, the study of human embryology was largely con-
fined to animal models of human development, such as
mouse (15,16) and nonhuman primate (17,18) ES cells.
Given the differences in developmental patterns among
species, and the ethical implications of in vivo work with
humans, the use of a human in vitro model is desirable for
the study of human development. The derivation and char-
UMAN EMBRYONIC STEM (hES) CELLS, like other stem
cells, have the capacity to self-renew without dif-
acterization of human embryonal carcinoma (hEC) cells in
the 1970s and 1980s was the first step in addressing the
need for a species specific model of human development
(19–23). Characterization of many hEC cell lines revealed
their capability to differentiate into numerous different cell
types; however, the existence of chromosomal abnormal-
ities limited their eventual use for clinical research in hu-
mans (22,24). The derivation and characterization of hES
cells and human embryonic germ (hEG) cells in 1998 al-
lowed for the emergence of a new model for the study of
human embryonic development and renewed hope for clin-
ical applications for tissues generated from human em-
bryonic cells in vitro (1,4,6,25).
Although hES cells have differentiated into many dif-
ferent tissue types in vitro (5,26–34), clinical uses and
specific models for human disease have not yet been re-
alized from the study of hES cells. One factor contribut-
Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, San Francisco,
CA 94143.

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ing to the slow pace of research with hES cells lies in the
difficulty of propagating and maintaining undifferenti-
ated hES cells in vitro. When hES cells are grown in sub-
optimal conditions, they spontaneously differentiated into
other cell types, thereby losing their self-renewal and
pluripotent qualities (1,4,6). Thus, hES cells require a
specialized growth environment to retain an undifferen-
tiated phenotype, and the components of this optimal
growth environment are constantly undergoing revisions
and improvements. The current state of research regard-
ing the growth and maintenance of hES cells is summa-
rized below, along with basic protocols utilized by this
laboratory for the propagation and characterization of
hES cells.
PROPERTIES OF UNDIFFERENTIATED
hES CELLS
Before addressing factors that can contribute to the
growth and maintenance of undifferentiated hES cells, it
is necessary to articulate the distinct qualities that make
it possible to distinguish between differentiated and
undifferentiated hES cells. Below, the properties of
undifferentiated hES cells, including cell and colony
morphology, cell-surface antigens, gene expression, and
pluripotency, are described briefly.
Maintaining hES cell morphology and density pro-
motes healthy pluripotent cell growth. Undifferentiated
hES cells have a distinct morphology when viewed un-
der the microscope (Fig. 2A,B) (1,4). Individual cells
within the colony have a large nucleus and prominent nu-
cleoli. The cells are tightly packed within the colony and
maintain a defined border at the periphery of the colony.
The morphology of an undifferentiated hES colony is
sharply contrasted by that of spontaneously differentiat-
ing hES colonies, which can take on many different forms
(Fig. 2C–H). For instance, differentiating colonies of hES
cells may appear to lose their tight border, and cells
within the colony may begin to enlarge, flatten, and sep-
arate (Fig. 2C–H). In other cases, differentiating hES cells
may pile up on one another within the colony, making it
BODNAR ET AL.
244
PERCENT b-ACTIN EXPRESSION
DAYS IN DIFFERENTIATION CULTURE
OCT-4
NCAM
FLK-1
AFP
FIG. 1.
on irradiated CF-1 feeders were harvested and cultured under differentiation conditions to form EBs, and harvested over a pe-
riod of 2 weeks. OCT-4, marker of undifferentiated ES cells; AFP, endoderm marker; FLK-1, mesoderm marker; NCAM, an ec-
toderm marker. These markers were assayed at each timepoint. Levels of the markers shown were compared with levels of b-
actin, a housekeeping gene, in the same reaction.
Semiquantitative RT-PCR analysis of hES cell gene expression during embryoid body differentiation. H9 cells cultured

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appear thick and opaque (Fig. 2C–D). Large differenti-
ating colonies can also form embryoid body-like struc-
tures and neural-like structures (Fig. 2E–F) or display
crater formation within the colony (Fig. 2C–D).
The profile of surface antigens displayed on undiffer-
entiated hES cells matches that of nonhuman primate ES
cells and hEC cells. It is well established that hES cells
express stage-specific embryonic antigen (SSEA)-3,
UNDIFFERENTIATED HUMAN ES CELLS
245
FIG. 2.
states of differentiation. The four photos in the left column are shown at 103 magnification, with a 203 magnification of the
same photo in the right column. (A,B) Undifferentiated hES cell colony pictured shows cells tightly packed within the colony,
and a defined border at the periphery of the colony, even when viewed at high magnification. (C,D) A differentiating colony
with cells piling up around the edge of the colony, and flattening and spreading out in the center of the colony. Differentiating
cells in the center of this colony are larger in size than undifferentiated hES cells. (E,F) Neurite-like processes extend from the
edge of the differentiating colony. This hES colony no longer exhibits a defined border on all sides, and the cells along the edges
of the colony have begun to enlarge and spread out. (G,H) The enlarged individual cells in the middle of this differentiating
colony are easily distinguished from one another, and no longer tightly packed together.
Examples of differentiated and undifferentiated hES cell morphology. HSF-6 hES cell colonies are shown in various
A
C
B
D
F
H
E
G

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SSEA-4, as well as TRA-1-60 and TRA-1-81, and down-
regulate these markers upon differentiation (Fig. 3)
(1,4,24,35–37). Unlike mouse embryonic stem (mES)
cells, undifferentiated hES cells do not express SSEA-1.
Undifferentiated hES cells also stain positively for alka-
line phosphatase (1,4,38), and demonstrate telomerase
activity (1,37,39).
The human gene OCT-4, encoding a POU-domain
transcription factor, is highly expressed in undifferenti-
ated hES cells and is necessary to maintain the pluripo-
tent state of ES cells (1,4,36,38,40). As hES cells differ-
entiate, OCT-4 gene expression decreases to low levels
(4,26,41). In addition, as OCT-4 expression decreases,
the expression of genes representative of the three em-
bryonic germ layers, such as AFP, FLK-1, and NCAM,
increase (Fig. 1). Other genes expressed in undifferenti-
ated hES cells, some of which may function in the main-
tenance of pluripotency, include CRIPTO/TDGF1 (42,
43), THY1 (42,43), FGF4 (36,42,43), REX1/ZFP42 (36,
42,43), SOX2 (36,42,43), and NANOG (44,45).
Human ES cells are pluripotent and therefore are ca-
pable of differentiating to cells of all three embryonic
germ layers in vivo and in vitro. When injected into im-
munodeficient mice, hES cells form teratomas in 1–4
months, demonstrating their potential to differentiate in
vivo (1,4,6,39). Teratomas may contain multiple complex
structures, including tissues from each of the embryonic
germ layers: endoderm, mesoderm, and ectoderm (Fig.
4). In vitro multilineage differentiation of hES cells can
be achieved through growth in adherent or suspension
culture without feeders. Human ES cells in suspension
culture without feeders differentiate to form tightly
packed balls of cells referred to as embryoid bodies (EBs)
(2,5,6,26,29,31,34), which contain many different cell
types, as indicated by reverse transcriptase (RT)-PCR
(Fig. 1) and histological (Fig. 4) analysis. Multilineage
differentiation of hES cells in vitro has also been achieved
through co-culture with various cell lines permissive to
differentiation, such as S17 (27), C166 (27), and END-2
(33).
Lastly, unlike mES (46–48) and hEC cells (49), aneu-
ploidy occurs rarely in hES cells. Human ES cells main-
tain a normal karyotype even after prolonged periods in
culture (1,4,38,39).
FACTORS CRUCIAL TO THE
MAINTENANCE OF HES CELLS
Five years after the first report of the derivation of hES
cell lines (1), the methods and techniques used to main-
tain hES cells in an undifferentiated proliferative state are
still undergoing revisions. According to current research,
several variables are thought to contribute to the growth
and differentiation in vitro, and these variables are dis-
cussed below.
Feeder cell layers
Human ES cells are routinely grown on a layer of
feeder cells. Historically, mitotically inactivated mouse
embryonic fibroblasts (MEFs) have been used success-
fully to support hES cell growth and maintenance
(1,4,39). Feeder cells are normally mitotically inactivated
using irradiation or through incubation with mitomycin
C. Human ES cells grown on feeders inactivated by ir-
radiation or mitomycin C seem morphologically similar,
and remain pluripotent in both cases. RT-PCR analysis
suggests that hES cells grown on mitomycin C-treated
feeders express similar levels of genes indicative of en-
doderm, mesoderm, and ectoderm differentiation (Fig. 5).
Although mitomycin C-treated feeders are able to main-
tain hES cells in an undifferentiated state, variability
among batches of cells treated with mitomycin C may
produce unusual gene expression patterns in hES cells
(our unpublished results). Recent developments also in-
dicate that certain human cell lines are capable of sup-
porting the growth and maintenance of undifferentiated
hES cells (our unpublished results, 50–53). The success-
ful mouse or human feeder layer is an important com-
ponent of hES cell culture in vitro, and has distinct char-
acteristics that are capable of influencing the growth and
differentiation of hES cells in several different capaci-
ties.
First, evidence suggests that feeders likely secrete sol-
uble factors into culture medium that are necessary for
maintenance of undifferentiated hES cells. Reports indi-
cate that when undifferentiated hES cells are grown in
the absence of a feeder cell layer, they differentiate un-
less conditioned medium from mouse fibroblast cultures
is used to supplement the hES culture medium (38). Al-
though the exact identities of the secreted factors neces-
sary for hES cell maintenance have not yet been identi-
fied, various candidate proteins have been identified in
medium conditioned by mouse fibroblast feeders (54).
Although some mES cell lines may be propagated in the
absence of a feeder layer when leukemia inhibitory fac-
tor (LIF) is added to the culture medium, LIF is insuffi-
cient to maintain hES cells in an undifferentiated state in
the absence of feeder cells (1,4). Recent reports indicate
that synthetic culture additives that activate the canoni-
cal Wnt pathway in hES cells (45) or the addition of a
combination of growth factors such as LIF, transforming
growth factor-b1 (TGF-b1), and basic fibroblast growth
factor (FGF-2) (55) may be sufficient to sustain undif-
ferentiated hES cells in the absence of feeders. Further
research and functional analysis of such proteins and
pharmacological agents may eventually elucidate mech-
anisms that affect signal transduction pathways, tran-
BODNAR ET AL.
246

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scription, growth, and differentiation in hES cells,
thereby leading to the commercial production of a de-
fined synthetic medium capable of supporting the growth
of undifferentiated hES cells. However, given the current
state of research, the use of mouse or human feeder cells
is the most widely used method of providing the soluble
factors necessary to support the growth and maintenance
of undifferentiated hES cells.
Cell–cell interactions between feeder layer and hES
cell colonies influence the growth and differentiation of
the hES cells. Human ES cells cultured in the absence of
feeder cells (1,4) or a purified extracellular matrix (38)
differentiate spontaneously. Although most hES cell lines
require contact with a living feeder layer of human or
mouse feeders, evidence suggests that some cell lines can
be maintained on a synthetic feeder layer with the addi-
tion of conditioned medium from living feeder cells. Xu
and colleagues (38) were able to sustain four lines of un-
differentiated hES colonies (H1, H7, H9, and H14) for a
prolonged period of time using a synthetic matrigel and
UNDIFFERENTIATED HUMAN ES CELLS
247
FIG. 3.
for 4 days, and then fixed and stained according to the “immunohistochemistry” protocol described above using Chemicon primary
antibodies, and FITC-conjugated secondary antibodies from Sigma. All photos were taken at 43 magnification. Bright-field pic-
tures of the fluorescently stained colonies are shown directly beneath the corresponding fluorescent image. (A) SSEA-1 antibody;
(B) TRA-1-60 antibody; (C) TRA-1-81 antibody; (D) SSEA-3 antibody; (E) SSEA-4 antibody.
Immunohistochemical staining of undifferentiated hES cells. HSF-6 cells were grown on irradiated CF-1 feeder cells
FIG. 4.
Hematoxylin & Eosin-stained paraffin section of a teratoma generated from HSF-6 hES cells in an immunodeficient beige mouse,
as described in the “In vivo differentiation to teratomas” protocol. (B) Hematoxylin & Eosin-stained paraffin section of an HSF-
6 embryoid body, differentiated for 14 days as described in the “In Vitro differentiation to embryoid bodies” protocol.
Human ES cell differentiation to teratomas and embryoid bodies. Both pictures are shown at 103 magnification. (A)
AB