Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro.
ABSTRACT Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called 'embryoid bodies', are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by 'gain of function' and 'loss of function' approaches in vitro.
- [show abstract] [hide abstract]
ABSTRACT: The establishment of embryonic cell lines from swine should be useful for studies of cell differentiation, developmental gene regulation and the production of transgenics. This paper summarizes the establishment of porcine (Sus scrofa) embryonic stem (ES) cell lines from preimplantation blastocysts and their ability to develop into normal chimaeras. ES cells can spontaneously differentiate into cystic embryoid bodies with ectodermal, endodermal, and mesodermal cell types. Further, culture of ES cells to confluence or induction of differentiation with retinoic acid or dimethylsulfoxide results in morphological differentiation into fibroblasts, adipocytes, and epithelial, neuronal, and muscle cells. These ES cells have a normal diploid complement of 38 chromosomes. Scanning electron microscopy of the ES cells reveals a rounded or polygonal, epithelial-like cell with numerous microvilli. The differentiation of these embryonic cell lines into several cell types indicates a pluripotent cell. Furthermore, chimaeric swine have been successfully produced using such ES cells.Reproduction Fertility and Development 02/1994; 6(5):563-8. · 2.58 Impact Factor
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ABSTRACT: Despite intense interest in understanding the differentiation of vascular smooth muscle, very little is known about the cellular and molecular mechanisms that control differentiation of this cell type. Progress in this field has been hampered by the lack of an inducible in vitro system for study of the early steps of smooth muscle differentiation. In this study, we describe a model system in which multipotential mouse P19 embryonal carcinoma cells (P19s) can be induced to express multiple characteristics of differentiated smooth muscle. Treatment of P19s with retinoic acid was associated with profound changes in cell morphology and with the appearance at high frequency of smooth muscle alpha-actin-positive cells that were absent or present at extremely low frequency in parental P19s. A clonal line derived from retinoic acid-treated P19s (9E11G) stably expressed multiple characteristics of differentiated smooth muscle, including smooth muscle-specific isoforms of alpha-actin and myosin heavy chain, as well as functional responses to the contractile agonists phenylephrine, angiotensin II, ATP, bradykinin, histamine, platelet-derived growth factor (PDGF)-AA, and PDGF-BB. Additionally, 9E11G cells expressed transcripts for MHox, a muscle homeobox gene expressed in smooth, cardiac, and skeletal muscles, but not the skeletal muscle-specific regulatory factors, MyoD and myogenin. Results demonstrate that retinoic acid treatment of multipotential P19 cells is associated with formation of cell lines that stably express multiple properties of differentiated smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)Circulation Research 06/1995; 76(5):742-9. · 11.86 Impact Factor
- Annals of the New York Academy of Sciences 04/1995; 752:460-9. · 4.38 Impact Factor
Cytotechnology 30: 211–226, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Embryonic stem cell differentiation models:
cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth
muscle cell differentiation in vitro
Kaomei Guan, Jürgen Rohwedel∗& Anna M. Wobus
“In Vitro Differentiation” Group, IPK Gatersleben, D-06466 Gatersleben, Germany
Received 14 April 1998; accepted 23 July 1998
Key words: cardiogenesis, cell differentiation, gene expression, mouse embryonic stem cells, myogenesis,
Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of
undifferentiatedcells. ES cells providean important cellular system in developmentalbiology for the manipulation
of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as
embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three
primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in
vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle,
neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion
channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern
developmentalpatternwas foundto be influencedbydifferentiationand growthfactormoleculesor by xenobiotics.
Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of
function’ approaches in vitro.
Abbreviations: ES cells – embryonic stem cells; EBs – embryoid bodies; ECC – embryonic carcinoma cells; FCS
– fetal calf serum; FCS-DCC – dextran-coated charcoal-treated FCS; RA – retinoic acid; VSM – vascular smooth
Permanent lines of totipotent mouse embryonic stem
biologyduringthe last years, since their establishment
from undifferentiated embryonic cells of blastocyst
stage embryos by Evans and Kaufman (1981) and
Martin (1981). ES cells injected into a host blastocyst
may be integrated into the inner cell mass and partic-
ipate in the embryonic development. After retransfer
of these blastocysts into pseudopregnant foster moth-
ers, the in vitro cultivated ‘donor’ ES cells are able to
∗Present address: Department of Medical Molecular Biology,
Medical University of Lübeck, D-23538 Lübeck, FRG
generate cells of all lineages including the germ line
and build up chimaeric animals in vivo (Bradley et al.,
1984). Therefore, ES cells provide an important cellu-
lar system for manipulating preselected genes in mice
by the gene targeting technology(Thomas and Capec-
chi, 1987). It enabled the generation of numerous
genetically manipulated ‘knock out’ mice (Brandon
et al., 1995), some of them serve as mouse models
for heritable human diseases that are attributable to
mutations at single genetic loci (Clarke, 1994).
Permanent ES cell lines are routinely cultivated
from the inner cell mass (ICM) of mouse blastocysts
(Evans and Kaufman, 1981; Martin, 1981; Wobus
et al., 1984; Figure 1), from single blastomeres of
8-cell-stages (Wobus et al., 1991) or morulae stage
Figure 1. ES cell technology in vitro. Permanent embryonic stem cell lines (ESC) were cultivated from the inner cell mass (ICM) of mouse
blastocysts, and embryonic germ cell lines (EGC) were cultivated from primordial germ cells (PGC). These pluripotent ESC or EGC are able
to differentiate via “embryoid bodies” (EBs) into derivatives of the endodermal, ectodermal and mesodermal lineages in vitro
embryos (Eistetter, 1989). Besides ES cells, two other
types of undifferentiated embryonic cells have been
established as permanent cell lines, the embryonal
carcinoma (EC) cells, malignant stem cells of terato-
carcinomas derived by extrauterine transfer of early
embryos (Martin and Evans, 1975; Stevens, 1984),
and the embryonic germ (EG) cells cultivated from
primordial germ (PG) cells (Figure 1; Resnick et al.,
1992; Stewart et al., 1994). Both totipotent cell types,
ES and EG cells, have been shown to participate in
normal development when injected into blastocysts
(Gardner and Brook, 1997).
ES, EG and EC cell lines exhibit characteristics of
undifferentiated embryonic cells in vitro: (i) pluripo-
tent differentiation capacity (Figure 1; Doetschman et
al., 1985; Keller, 1995; Rohwedel et al., 1994, 1996;
Maltsev et al., 1993, 1994; Strübing et al., 1995; Bain
et al., 1995; Dani et al., 1997; Bagutti et al., 1996;
Risau et al., 1988; Drab et al., 1997; Wobus et al.,
1991, 1994b, 1997a; Wobus and Guan, 1998), expres-
sion of (ii) endogenous alkaline phosphatase (Resnick
et al., 1992), (iii) stage-specific embryonic antigen
SSEA-1 (Solter and Knowles, 1978; Resnick et al.,
1992), and (iv) germline-specific transcription factor
Oct-4 (Schöler et al., 1990), (v) hypomethylation of
DNA (Monk, 1990), and (vi) a short G1phase of the
cell cycle (Rohwedel et al., 1996).
To mimic the differentiation of totipotent stem
cells in the embryo, in vitro cultivated ES cells were
differentiated as embryo-like aggregates, so-called
‘embryoid bodies’ (EBs). Within these EBs, cellu-
lar derivatives of all three primary germ layers of
endodermal, ectodermal and mesodermal origin are
differentiated. The pluripotent/totipotent ES cell lines
develop from an undifferentiated stage resembling
cells of the early embryo into terminally differenti-
ated stages of the cardiogenic (Wobus et al., 1991,
1997a, 1997b; Maltsev et al., 1993, 1994; Hescheler
et al., 1997; Miller-Hance et al., 1993; Wobus and
Guan, 1998), myogenic (Miller-Hance et al., 1993;
Rohwedel et al., 1994; Rose et al., 1994), neurogenic
(Strübing et al., 1995; Bain et al., 1995; Fraichard et
al., 1995; Okabe et al., 1996), hematopoietic (Keller,
1995; Wiles and Keller, 1991; Hole and Smith, 1994)
or adipogenic (Dani et al., 1997) lineage, as well as
into endodermal (Sauer, 1998), epithelial (Bagutti et
al., 1996), endothelial (Risau et al., 1988), vascular
smooth muscle (VSM, Risau et al., 1988; Weitzer et
al., 1995; Drab et al., 1997) and chondrogenic cells
(Rohwedel, unpublished data).
We found that the terminally differentiated cells
showed pharmacological and physiological properties
of specialized cells: in vitro differentiated cardiomy-
ocytes resemble characteristics of atrial-, ventricle-,
purkinje- and pacemaker-like cells (Maltsev et al.,
1993, 1994; Wobus et al., 1997b; Hescheler et al.,
1997), neuronal cells are characterized by inhibitory
and excitatory synapses (Strübing et al., 1995; Okabe
et al., 1996),andcardiac, myogenic,neuronalandvas-
and tissue-specific functional receptors (Wobus et al.,
1991; Strübing et al., 1995; Rohwedel et al., 1998a;
Drab et al., 1997; Wobus et al., 1997b). Further-
more, the interaction of neuronal and skeletal muscle
cells resulted in the formation of postsynaptic-like
membranes (Rohwedel et al., 1998a).
In the following review, the ES cell differenti-
ation systems of cardiogenesis, myogenesis, neuro-
genesis, epithelial and VSM cell differentiation are
summarized with respect to the expression pattern of
genes, proteins and to their functional properties (for
haematopoietic differentiation, see Keller, 1995, for
adipogenic differentiation, see Dani et al., 1997). Fur-
thermore, a short overview about the possibilities to
modulate the in vitro differentiation pattern by exoge-
nous compounds, and to use ES cells for ‘gain of
function’ and ‘loss of function’ approaches are given.
Material and methods
Culture of undifferentiated ES cells and embryoid
ES cells of lines D3 (Doetschman et al., 1985), R1
(Nagy et al., 1993) or CCE (Wiles and Keller, 1991),
were cultivated on a feeder layer of primary mouse
embryonic fibroblasts (Wobus et al., 1991) in Dul-
becco’s modified Eagle’s medium (DMEM, Gibco
BRL, Life Technologies, Eggenstein, Germany) sup-
plemented with 15% heat-inactivated fetal calf serum
(FCS, selected batches, Gibco), L-glutamine (2 mM,
Gibco), β-mercaptoethanol (β-ME, final concentra-
tion 5×10−5M; Serva, Heidelberg, Germany) and
non-essential amino acids (NEAA, stock solution di-
luted 1:100, Gibco) as described (Wobus et al., 1991;
Maltsev et al., 1993) to keep ES cells in the undiffer-
In addition, ES cell lines may be grown with-
out feeder layer in media supplemented with 10–
Figure 2. ES cell differentiation protocol. ES cells (ESC) are routinely cultivated on mouse embryonic feeder layer to keep the cells in the
undifferentiated stage (a). For in vitro differentiation, ES cells were cultivated as embryoid bodies (EBs) in hanging drops for two days,
and after suspension culture for 3–5 days. EBs are plated between days 5–7, depending on the differentiation lineage. EBs attach to tissue
culture plates and differentiated cells develop in the EB outgrowths. Shown are the quantitative estimations of ES cell differentiation into
spontaneously beating cardiac cells (b), skeletal muscle cells (c), neuronal cells (d) without (open symbols) and with induction by retinoic
acid (RA; filled symbols), endodermal/ epithelial cells (e) and vascular smooth muscle cells (f) without (open symbols) and with induction by
RA and dibutyryl-cAMP (db-cAMP; filled symbols). The cells were characterized by morphological analysis of EBs, i.e., the number of EBs
containing differentiated cells was estimated as percentage.
for preparation, see Rohwedel et al., 1996) for growth
in the undifferentiatedstage, or on both, a feeder layer
and LIF-supplemented media.
The ES cell differentiationprotocols shown in Fig-
ure 2 have been described in detail (see Wobus et al.,
1991, 1997a). In principal, for the development of
ES cells into differentiated phenotypes, the pluripo-
tent cells were cultivatedas EBs by the ‘hangingdrop’
method (Wobus et al., 1991; Rudnicki and McBurney,
1987) or by ‘mass culture’ (Doetschman et al., 1985).
After plating the EBs at day 5 or 7, the differentiated
cells are grown out and the number of EB outgrowths
withthespecific differentiatedcelltypewas calculated
as a percentage(Figure 2b–f). The process of morpho-
logical differentiation in the EBs is accompanied by
changes in the pattern of gene expression as well as
protein formation, development of ion channels and
tissue-specific receptors (Figure 2b–f, Figure 3b–f).
The different ES cell lines show pluripotent devel-
opmental capacities in vitro, i.e., they differentiate in
the EB outgrowths into many differentiated cell types.
But, to obtain maximaldifferentiationof a defined cell
type, specific cell lines andcultivationconditionswere
Figure 3. Immunocytological analysis of tissue-specific proteins. a) Undifferentiated ES cells of line D3 immunostained with a monoclonal
antibody against the stage-specific embryonic antigen, SSEA-1, and ES cell-derived differentiated cellular phenotypes are shown (b–f). Car-
diomyocytes are immunostained by monoclonal antibodies against titin (Z-band; b), myotubes by nebulin (c), neuronal cells by neurofilament
proteins NF160 kDa (d), epithelial cells by cytokeratin K19 (e) and VSM cells by smooth muscle α-actin (f), respectively. Undifferentiated ES
cells immunostained by SSEA-1 (a) were scanned in single sections by the confocal laser scanning microscope, EB outgrowths immunostained
for cytokeratin 19 (e), and smooth muscle α-actin-positive cells (f) were scanned in 8 sections (1 µm; e) and 4 sections (0.5 µm; f), respectively.
Bars represent 10 µm.
Detection of tissue-specific genes by
semi-quantitative RT-PCR analysis
The expression of tissue-specific genes in EBs and
outgrowths is analyzed by semi-quantitative RT-PCR
using the “primer-dropping” method according to
Wong et al. (1994) as described (Wobus et al., 1997b).
EBs or outgrowths were collected at several stages af-
terplatingatday5 or7. ThetotalRNA was isolatedby
the single step extraction method according to Chom-
czynski and Sacchi (1987) and mRNA was reverse
transcribed using Oligo d(T)16primer (Perkin-Elmer,
Überlingen, Germany) and amplified using oligonu-
genes (see Wobus et al., submitted).
Expression of tissue-specific proteins analyzed by
The formation of tissue-specific proteins in EB out-
growths is analyzed by immunofluorescence analysis.
EBs plated on cover slips were rinsed twice with
PBS, fixed with methanol: acetone (7:3) at –20◦C
for 10 min and processed for immunofluorescence
microscopy (Maltsev et al., 1993). Antibodies for
the analysis of tissue-specific proteins are, for ex-
ample, the titin (Z-band)-specific mAb T12 (Fürst et
al., 1988) for cardiomyocytes and skeletal myocytes
(a cardiomyocyte is shown in Figure 3b), a nebulin-
specific mAb Nb2 (Rohwedel et al., 1998a) for skele-
tal muscle cells (Figure 3c), a neurofilament protein
neuronal cells (Figure 3d), a cytokeratin K19-specific
mAb (TROMA III, a gift of Dr. Kemler, Freiburg) for
endodermaland epithelialcells (Figure3e; see Bagutti
et al., 1996) and a smooth muscle α-actin-specific
mAb 1A4 for vascularsmooth muscle cells (Figure 3f;
see Drab et al., 1997).
Cardiomyocytes were isolated as single beating
cells from EB outgrowths (see Maltsev et al., 1993;
1994) and were used for immunostaining (Figure 3b)
and for the characterization of action potentials and
ion channels by patch-clamp analysis.
Pharmacological and physiological analyses
To demonstrate the functional properties of in vitro
differentiated excitable cardiac, skeletal muscle, neu-
ronal or VSM cells, electrophysiological and pharma-
cological techniques were employed. The expression
of ion channels on ES cell differentiated cardiomy-
ocytes (Maltsev et al., 1993; 1994), skeletal muscle
(Rohwedel et al., 1994), neuronal (Strübing et al.,
1995; Fraichard et al., 1995) and VSM cells (Drab
et al., 1997) was analyzed by the whole-cell config-
uration of the patch-clamp technique (Hamill et al.,
1981). Action potentials of isolated cardiomyocytes
were analyzed by the same technique (Maltsev et al.,
1993; Fässler et al., 1996; Hescheler et al., 1997).
The spontaneous beating capacity of ES and EC
cell-derived cardiomyocytes enabled to investigate
chronotropic effects of cardioactive substances by
measuring the beating frequencies of cardiac cells
(Wobus et al., 1991, 1994b). An inverted microscope
Diaphot-TMD (Nikon) equipped with a 37◦C and
5% CO2incubation chamber was used. Cardioactive
drugs were cumulatively added to pulsating clusters
of EB outgrowths, the frequencies were measured
from control (= basal level of beating frequency)
and agonist-treated variants to make up dose-response
curves. By using the LUZIA imaging device (Nikon),
a semi-automatic computer-assisted imaging system
for a routine screening of cardioactive drugs was
established(Pich etal., 1997;Wobusetal., submitted).
The receptor activity of ES cell-derived VSM cells
was analyzed by measuring the increase of intracel-
lular [Ca2+] transients by confocal laser scanning
microscopy of fluo-3-labelled VSM cells. Vasoactive
agonists were able to evoke intracellular free [Ca2+]
transients in VSM cells differentiated from ES cells
(Drab et al., 1997).
ES cells develop into differentiated phenotypes
Depending on the specific differentiated cellular phe-
notypes, distinct protocols with different ES cell lines
were established. The ‘hanging drop’ method gen-
erated EBs of a defined cell number and size. This
technique has been used for developmentalstudies be-
cause the differentiation pattern is dependent on the
numberofES cells which differentiatewithin the EBs.
It was found that the following variables influenced
the developmental potency of ES cells in culture: (1)
quality of fetal calf serum, growth factors and medium
additives, (3) ES cell lines used, and (4) the time of
Mouse ES cells can differentiate into cardiogenic,
myogenic, neuronal-, epithelial- and vascular smooth
muscle-like cells in vitro, and the expression of tissue-
specific genes, proteins and ion channels is devel-
opmentally controlled during differentiation. In the
following chapter a short overview about the ES cell-
derived phenotypes is given with respect to tissue-
specific genes, proteins and ion channels (Figure 4).
ES cells of lines D3, R1 and CCE differentiated via
EBs into clusters of spontaneously beating cardiomy-
ocytes. Optimal cardiac differentiation was achieved
by using 400 cells of line D3 or R1 for the prepa-
ration of EBs. We used different media for EB de-
velopment and cellular differentiation: DMEM sup-
plemented with 20% FCS, L-glutamine, β-ME and
NEAA (‘DMEM differentiation medium’), or Is-
cove’s modification of DMEM (IMDM, Gibco) sup-
plementedwith 20%FCS, L-glutamine,NEAA andα-
monothioglycerol 3-mercapto-1,2-propandiol (MTG,
final concentration 450 µM; Sigma; ‘IMDM differ-
entiation medium’). EBs were plated between days 5
First beating clusters in EBs can already be seen
in 7 day old EBs or 1 to 2 days after plating (Wobus
et al., 1991; Maltsev et al., 1993, 1994), but maximal
differentiation of beating cardiomyocytes is achieved
several days (7 to 10 d) after EB plating at days 5 to 7
At the level of gene expression, a characteristic se-
quence of cardiac-specific gene expression was found.
The cardiac-specific transcription factor Nkx 2.5 was
weakly expressed in ES cells of line D3 and in EBs at
day 3, but maximally in EBs between days 5 and 5 +
12 (Wobus and Guan, 1998). The gene encoding the
α1subunit of the L-type Ca2+channel (α1CaCh) was
first expressed in EBs two days before beating cells
appeared, followed by α- and β-cardiac myosin heavy
chain (MHC) around the day when first beating cells
were observed.Finally, genes expressed in specialized
cardiac cell types, such as those encoding atrial natri-
uretic factor (ANF, expressed mainly in atrial cells)
and myosin light chain isoform 2v (MLC-2v, spe-
cific for ventricular cells; Fässler et al., 1996; Wobus
et al., 1997b; Hescheler et al., 1997) were detected
Cardiomyocytes differentiated from EB out-
growths revealed a specific sequence of expression of
the sarcomeric proteins during cardiogenesis as fol-
lows: titin(Z-band-specific;Figure3b),α-actinin, my-
omesin, titin (M-band-specific), sarcomeric MHC and
α-actin in early cardiomyocytes. M-protein was only
expressed at terminal differentiation stages (Figure 4;
Wobus and Guan, 1998).
During differentiation, ES cells developed into
which show pacemaker-like action potentials at the
early stage (day 7). They further specialize into atrial-
(38%), ventricular- (48%) and sinusnodal-like (14%)
cells, based on electrophysiological determination of
action potentials at the terminal differentiation stage
(between days 14 to 26 after plating; Maltsev et
al., 1993; Fässler et al., 1996). Recently, a fourth
type of action potential was described that repre-
sented Purkinje-like cells (Hescheler et al., 1997;
Wobus et al., 1997b). The various types of action
velopmental stages correlated with the expression of
specialized types of ion channels (Figure 4; Malt-
sev et al., 1994; Hescheler et al., 1997; Wobus and
Guan, 1998). Recently (Maltsev et al., submitted),
the modulation of ICa was used as a functional as-
say to test different components of the β-adrenergic
signaling cascade during cardiomyocytedevelopment.
Maltsev et al. found that the uncoupling and/or low
expression of Gs-protein accounted for the ICa in-
sensitivity to β-adrenergic stimulation in very early-
developmental stage cardiomyocytes. But in early-
developmental stage cells, the uncoupling is due, at
least in part, to a high intrinsic activity of phospho-
diesterases. These results together with previous data
(Maltsev et al., 1993, 1994)indicatethe normalcourse
of development for ES cell-derived cardiomyocytes
similar to embryonic cardiomyocytes. Furthermore,
we found that the differentiated cardiomyocytes re-
sponded with characteristic chronotropic responses
to cardiotropic drugs comparable to cardiomyocytes
from living organisms (Wobus et al., 1991; Pich et al.,
Differentiation of ES cells into skeletal muscle cells
was shown by the formation of myoblasts which fused
into multinucleated myotubes during terminal differ-
entiation. Optimal development into skeletal muscle
cells was achieved by using 800 cells of line D3
(Rohwedel et al., 1998a) or 600 cells of line BLC6
(Rohwedel et al., 1994) in DMEM containing 15%
dextran-coatedcharcoal -treated FCS (DCC-FCS, Ro-
hwedel et al., 1998), L-glutamine, NEAA, β-ME,
sodium selenite (final concentration: 3 × 10−8M;
Sigma), bovine serum albumine (BSA, final concen-
tration: 0.1875%; Gibco) and transferrin (final con-
Figure4. Developmentally controlled expression of tissue-specific genes, proteins and ion channels after differentiation of EScells into cardiac,
skeletal muscle, neuronal, epithelial and VSM cells in vitro. Cardiogenesis: Genes encoding the cardiac transcription factor Nkx 2.5, the α1
subunit of the L-type Ca2+channel (α1CaCh), α- and β-cardiac myosin heavy chain (α-, β-MHC), atrial natriuretic factor (ANF) and the
ventricular isoform 2 of myosin light chain (MLC-2v) were analyzed by RT-PCR. The expression of sarcomeric proteins was determined by
immunofluorescence using the antibodies against: titin (Z-band), nonmuscle α-actinin, myomesin, sarcomeric MHC, α-actin and M-protein.
The cardiac-specific ion currents ICa(L-type Ca2+current), Ik,to(transient K+current), IK,ATP(ATP-modulated K+current), IK(outwardly
rectifying K+current), INa(inward Na+current), If(hyperpolarization-activated pacemaker current), IK1(inwardly rectifying K+current),
IK,ACh(muscarinic acetylcholine-activated K+current) were found during ES cell-derived cardiogenesis. Myogenesis: Myogenic determi-
nation genes myf5, myogenin, MyoD and myf6, the muscle-specific cell adhesion molecule M-cadherin, and the γ- and ?-subunit of the
nicotinic acetylcholine receptor (nAChR) were analyzed. The myocytes expressed muscle-specific proteins such as titin, nebulin, M-cadherin,
myogenin, sarcomeric MHC and slow C-protein, the latter only found to be expressed at terminal stages. Skeletal muscle-specific L-type
and T-type Ca2+channels (ICa,L; ICa,T) and functional nicotinic acetylcholine receptor-operated channels (nAChR) were expressed during
ES cell-derived myogenesis. Neurogenesis: A developmentally controlled expression pattern of genes encoding 68 kDa (NFL), 160 kDa
(NFM) and 200 kDa (NFH) neurofilament proteins, coding for the synaptic vesicle protein synaptophysin, the neuron-specific proteoglycan
neurocan, and the embryonic splice variant of the microtubule-associated protein tau was found during ES cell-derived neurogenesis. Neuron-
and glial cell-specific proteins NFL, NFM, NFH, SNAP-25, synaptophysin and glial fibrillary acidic protein (GFAP) were determinated by
immunofluorescence studies. Neuron-specific voltage-dependent ion currents: K+current (IK), Na+current (INa) and Ca2+current (ICa),
and neuron-specific receptors: γ-aminobutyric acid (GABAA), Glycin (Gly), Kainate (Kai) and N-methyl-D-aspartate (NMDA) were found in
neuronal cells by electrophysiological analyses. Epithelial differentiation: Epithelial cells expressed cytokeratins 8, 18, 19 (K8, K18 and K19)
at early stages, and, cytokeratins K8, K18, K10, K14 and involucrin (inv) at later stages. Vascular smooth muscle (VSM) cell differentiation:
VSM cells derived from ES cells preponderantly expressed vascular smooth muscle myosin heavy chain A (V-SM-MHC-A) and smooth muscle
α-actin (SM-α-Actin), the intestinal smooth muscle myosin heavy chain B (I-SM-MHC-B) was only slightly expressed at later stages. Three
voltage-sensitive ion channels, the smooth muscle-specific Ca2+channel (IK,Ca), the calcium-activated maxi K+channel (IK,Ca), and delayed
rectifying K+channel (IKv) were expressed in VSM cells. Receptors for angiotensin II (AII-R), bradykinin, histamin, platelet-derived growth
factor AB (PDGF AB), thrombin and endothelin-1 were functionally expressed.
centration: 0.01 mg/ml; Gibco) (‘DMEM-DCC dif-
ferentiation medium’), or in ‘IMDM differentiation
medium’. EBs were plated at day 5.
The first myoblasts/ myocytes appeared 4 (line
BLC6) or 5 to 7 days (D3) after EB plating (Fig-
ure 2b). Skeletal muscle cells fused into myotubes in
the EB outgrowths 1 or 2 days later (Figure 3c). Dur-
ing myogenic development, muscle-specific genes,
proteins and ion channels were time-dependently ex-
pressed as summarized in Figure 4.
During ES cell differentiation, genes encoding the
myogenic regulatory factors Myf5, myogenin, MyoD
and Myf6, the muscle-specific cell adhesion mole-
cule M-cadherin as well as the γ- and ?-subunit of
the nicotinic acetylcholine receptor (nAChR) were
expressed in a sequence closely resembling myoge-
nesis in vivo (Rohwedel et al., 1994; Rose et al.,
1994; Rohwedel et al., 1998a). The myogenic cells
expressed muscle-specific proteins such as sarcom-
eric MHC, myogenin and M-cadherin (Rohwedel et
al., 1994; Rohwedel et al., 1995) as well as titin,
nebulin (Rohwedel et al., 1998a), α-actinin and slow
C-protein (Guan, unpublished). In addition, electro-
physiological studies using the patch-clamp technique
demonstrated the expression of functional nicotinic
cholinoceptors and T-type Ca2+channels with de-
creasing density, as well as L-type Ca2+channels
with increasing density on ES cell-derived muscle
cells comparable to developing muscle cells in vivo
(Figure 4; Rohwedel et al., 1994). Interactions be-
tween muscle cells and neuronal cells which both
differentiate in the same EBs resulted in the formation
of neuromuscular junctions. We demonstrated that
multinucleated contracting myotubes which appear at
a terminal differentiation stage formed postsynaptic-
like membranes exhibiting a clustering of nAChRs
colocalized with agrin and synaptophysin. A coex-
pression of the myogenic regulatory gene Myf6 and
the AChR ?-subunit gene was found, which paralleled
their expressionin adult muscle cells (Rohwedel et al.,
Different ES cell lines and protocols were established
for efficient differentiation of neuronal and glial cells
(Strübing et al., 1995; Bain et al., 1995; Fraichard et
al., 1995; Okabe et al., 1996).
D3 cells cultivated by the hangingdrop method for
two days followed by suspension culture in the pres-
enceof 10−7M retinoicacid (RA) between days 2 and
5 and plating at day 7 resulted in efficient neuronal
differentiation (Wobus et al., 1994a). In addition, EBs
prepared from 400 cells of line BLC6 cultivated in
DMEM-DCC differentiation medium in the presence
of 10−7M RA during the first two days of EB culture
and plating at day 4 showed an efficient differentiation
of functional neuronal cells (Strübing et al., 1995).
The frequency of spontaneous differentiation of
BLC6 cells into neuronalcells amountedto 15to 30%.
After differentiation induction by RA (Strübing et al.,
1995), the differentiation rate of neuronal cells was
increased to nearly 100% of the EBs (Figure 2d).
ES cells of line BLC6 differentiated after RA-
induction into neuronal cells which expressed neuron-
specific genes and were characterized by the complex
electrophysiological and immunocytochemical prop-
erties of postmitotic nervecells (Strübinget al., 1995).
Similar to ES cell-derived cardiac and myogenic dif-
ferentiation, also neurogenic differentiation preceeds
from progenitor cells to specialized cell types reflect-
ing a characteristic sequence of expression of neuron-
specific genes, proteins and ion-channels (Figure 3d;
Figure 4). Genes encoding the low (NFL) and middle
(NFM) molecularmass neurofilamentproteins and the
at an early stage of ES cell- derived neurogenesis(Ro-
hwedel et al., 1998a) in parallel to the expression
of voltage-gated ion channels, Ca2+, Na+and K+
channels (Strübing et al., 1995). Further differentia-
tion was characterized by an increase in the density
of voltage-gated ion channels, the onset of expres-
sion of receptor-operated ion channels in parallel to
the expression of genes and proteins characteristic for
matureneurons(Strübinget al., 1995; Rohwedelet al.,
In addition to the expression of specific neuronal
receptors, neuronal cells generated Na+-driven action
potentials and were functionallycoupled by inhibitory
(GABAergic) and excitatory (glutamatergic) synapses
as revealed by measurements of postsynaptic currents
(Figure 4; Strübing et al., 1995; Wobus et al., 1997a).
Bain et al. (1995) used a four-day mass culture
of ES cells to prepare EBs followed by a four-day
suspension culture in the presence of 5 × 10−7M
RA and plating at day 8. Another method described
differentiation factors (insulin, transferrin, selenium),
growth factors (bFGF) and extracellular matrix pro-
teins (fibronectin, laminin, polyornithine) as efficient
neuronal differentiation inducers (Okabe et al., 1996).
Epithelial cell differentiation
Differentiation of ES cells as EBs cultivated by the
hanging drop or the mass culture method in DMEM
or IMDM differentiation medium resulted in epithe-
genes and proteins (Bagutti et al., 1996).
Epithelial-like cells are one of the most prominent
cell types in the ES cell-derived EB outgrowths which
were found in all EB outgrowths without any spe-
cial differentiation induction (Figure 2d; Figure 3e).
Epithelial-specific genes and proteins characteristic
for early (cytokeratins K8, K18, K19), intermediate
(cytokeratin K14) and terminal (involucrin)differenti-
ation were expressed during EB development in vitro
(Figure 4; Bagutti et al., 1996).
Vascular smooth muscle (VSM) cell differentiation
A complex cell type which differentiated from ES
cells, are spontaneously contracting VSM cells. We
established a specific differentiation protocol by us-
ing RA and db-cAMP for the induction of VSM cells
during EB differentiation (Drab et al., 1997). For dif-
ferentiation of smooth muscle cells, ES cells of line
D3 were cultivated in hanging drops for 2 days and
after suspension culture for five days. EBs were plated
at day 7 followed by treatment with 10−8M RA and
0.5 × 10−3M db-cAMP between days 7 and 11 (Drab
et al., 1997). During differentiation of the EB out-
growths, the medium was changed every second day
and the first spontaneously contractingsmooth muscle
cells appeared around day 14 after plating.
Addition of RA and db-cAMP increased the dif-
ferentiation rate of VSM cells from a control level of
only 5–10% to more than 60% of the EBs 28 days
after plating (Figure 2e; Drab et al., 1997). During
ES cell differentiation in vitro, VSM-specific MHC
was preponderantlyexpressed between days 7 and 14,
which is in temporal agreement with the observation
in the dorsal aorta on day 10 postcoitum in vivo (Mi-
ano et al., 1994). The intestinal splice variant of the
smooth muscle MHC was only sligthly expressed at
a rather terminal stage of EB differentiation (Drab et
al., 1997). In addition, three distinct voltage-sensitive
ion channels: the calcium-activated maxi K+channel,
(IKca) the “delayed rectifier” K+channel (IKv) and
the dihydropyridine-sensitive (L-type) Ca2+channel
(ICa) were expressed in VSM cells differentiated from
ES cells (Drab et al., 1997). ES cell-derived VSM
cells were functionally characterized by [Ca2+] tran-
sients in response to the VSM cell-specific agonists
angiotensin II, bradykinin, histamine, endothelin-1,
PDGF AB, thrombin and vasopressin with an in-
creased intracellular Ca2+release (Figure 4; Drab et
AnotherprotocolforVSM cell differentiationused
ES cells of lines AB1 and AB2.1 cultivated as EBs in
by 15% FCS and 2 mM glutamine) for 4.5 days.
After plating of the EBs, the medium was partially
exchanged by fresh medium every third day (Weitzer
et al., 1995).
Modulation of differentiation
Modulation of differentiation by retinoic acid (RA)
The establishment of the ES cell differentiation
models allowed the study of cellular differentiation
processes duringembryonicdevelopmentin vitro. The
systems permitted the analysis of undifferentiatedem-
bryonic cells via progenitor cells into highly differ-
entiated and specialized cells of the cardiovascular,
myogenic and neurogenic lineages.
The controlled developmental process in the EBs
in vitro offered the possibility of modulating the de-
velopmental pattern by differentiation factors, growth
factors, or extracellular matrix (ECM) proteins. One
of the most effective differentiation factors is retinoic
acid (RA) which influenced time- and concentration-
dependently the differentiation of EC and ES cells
into cardiogenic (Wobus et al., 1994a, 1997b), myo-
genic (Wobus et al., 1994a), neurogenic (Wobus et al.,
1994a;Strübingetal., 1995,Bainet al., 1995)orVSM
cell (Drab et al., 1997) lineages.
It has been clearly shown that RA exerts its spe-
cific differentiation-inducing effect on ES cells in a
concentration- and time-dependent manner during EB
development. Treatment with high concentrations of
RA (10−7M) during the first 2 days of EB differ-
entiation increased the differentiation frequency of
neuronal cells and accelerated the neuronal differen-
tiation without changing the functional cell fates of
the differentiating neurons (Strübing et al., 1995), but
significantly inhibited cardiac differentiation (Wobus
et al., 1994a). Incubation of EBs with RA (10−8and
10−7M) between days 2 and 5 of EB development
genesis, but in an inhibition of cardiogenesis (Wobus
et al., 1994a). The RA-induced skeletal myocytes
functionally expressed tissue-specific Ca2+channels
and nicotinic cholinoceptors. In contrast, treatment
ning on day 5 resulted in an increased and accelerated
differentiation into the cardiogenic lineage and es-
pecially into ventricular cells (Wobus et al., 1994a
and 1997b). RA, both in the all-trans and in the 9-
cis configuration, accelerated cardiac differentiation
throughinductionof expressionof the cardiac-specific
α-cardiac MHC and MLC-2v genes, events which
resulted in an enhanced development of ventricular
cardiomyocytes (Wobus et al., 1997b).
RA treatment(10−8MRA incombinationwith0.5
× 10−3M db-cAMP) between days 7 and 11 of EB
development induced the differentiation of VSM cells
(Drab et al., 1997). Smooth muscle cells were fully
differentiated and expressed vascular-specific genes,
proteins, ion channels and receptors (Drab et al.,
The mechanisms whereby RA induces differenti-
ation are not known. However, as shown by various
groups (reviewed by Marshall et al., 1996), RA ex-
erts a wide variety of profound effects on vertebrate
development and cellular differentiation by activation
of homoeotic genes and other transcription factors.
Hogan et al. (1992) described the endogenous synthe-
sis of RA in Hensen’s node during a defined time win-
dow of embryonicdevelopmentin chicken. Therefore,
RA was suspected to be one of the most important
morphogens during vertebrate embryogenesis.
In vitro, RA induced MHox expression during the
differentiation of smooth muscle cells (Blank et al.,
3 (Jacob et al., 1997), MASH-1, MATH-1, neuroD
and NSCL-2 (Itoh et al., 1997) during neuronal differ-
entiation of mouse P19 cells. Furthermore, retinoids
promoted terminal muscle differentiation via activa-
tion of the muscle-specific myoD gene family (myoD,
myogenin, myf-5 and MRF-4) of transcription fac-
tors (Muscat et al., 1995). A novel RA-inducible gene
of the basic HLH family, Stra13, was found to be
expressed during mouse embryogenesis in neuroec-
toderm and in several mesodermal and endodermal
derivatives. Overexpression of Stra13 resulted in neu-
ronal differentiation of P19 cells, which without RA
induction undergo mesodermal and endodermal dif-
ferentiation indicating that Stra13 might be one of the
earliest RA target genes (Boudjelal et al., 1998).
With respect to cardiogenesis, homoeotic genes
and transcription factors, such as the tinman-related
Nkx 2.5 (Lints et al., 1993), the muscle-specific
MEF2C (Lin et al., 1997), the cardiac-specific
dHAND (Srivastava et al., 1995) or GATA4 genes
(Molkentin et al., 1997) might be candidate RA-
Modulation of differentiation by ‘gain of function’
Overexpression of a tissue-specific gene which is spa-
tially and temporally regulated during development
helps to reveal its function. Since the work of Palmiter
et al. (1982), ‘gain of function’ studies have been suc-
cessfully carried out by generating transgenic animals
carrying genes under the control of inducible or con-
the overexpression of genes during in vitro differenti-
ation of ES cells (Figure 5). We used the transcription
factor M-twist, a negative regulator of muscle differ-
entiation in a ‘gain of function’ approach. ES cell
clones stably transfected with the M-twist cDNA un-
der the control of a modified SV40 promoter showed
delayed differentiation of myogenic cells and skele-
tal muscle-specific gene expression depending on the
level of exogenous M-twist expression (Rohwedel et
al., 1995). Using the same approach, overexpression
of the homeobox gene HOXB4 during ES cell differ-
entiation in vitro resulted in enhanced differentiation
of erythroid progenitor cells (Helgason et al., 1996).
Furthermore, it was demonstrated that ES cells which
constitutively express the myogenic regulatory factor
MyoD preferentially differentiated into the myogenic
lineage (Dinsmore et al., 1996). Thus, in vitro ‘gain of
function’ studies using ES cells provide an alternative
strategy to transgenic animals.
Modulation of differentiation by ‘loss of function’
The establishment of optimal conditions for the devel-
opment of ES cells in vitro into cardiac, myogenic,
neuronal,epithelial and VSM cells enabledus to study
differentiation of genetically altered ES cells. After
gene inactivation by homologous recombination, ES
cells can be retransferred into blastocysts to regen-
erate chimaeric mice in vivo (Thomas and Capecchi,
1987). Null mutations could lead to homozygous ani-
mals whichshow(i)no mutantphenotypes,(ii) mutant
phenotypes, or (iii) result in embryonic lethality (Fig-
ure 5). In cases, in which the mutation leads to early
embryonic death, the differentiation of targeted ES
cells via EBs in vitro represents a new supplemen-
tary technique to analyze the effects of the specific
mutation on cellular differentiation (Figure 5).
We successfullyappliedthis approachtothe analy-
sis of loss of β1integrin functionon differentiation. In
vivo, a lack of β1integrin resulted in embryonic death
shortly after implantation (Fässler and Meyer, 1995).
By using the ES cell differentiation approach in vitro,
we showed that cell-matrix interaction via β1integrin
Figure 5. Modulation of embryonic differentiation by “gain of function” or “loss of function” strategies in vitro and the results of gene targeting
experiments in vivo (for explanation, see text).
is important for normal cardiogenesis, myogenesis,
neurogenesis, epithelial and VSM cell differentiation.
We found that during in vitro differentiation of β1-
null ES cells, the development into cardiomyocytes,
skeletal muscle and epithelial cells was severely im-
paired (Fässler et al., 1996; Bagutti et al., 1996;
Rohwedel, et al., 1998b; our unpublished data). In
general, mesodermal cell types such as cardiac and
myogenic cells developed with a delay, whereas dif-
ferentiation of neuronal cells was accelerated in the
absence of β1integrin. Usually, ES cells differenti-
ated into cardiomyocytes of atrial-, ventricular- and
pacemaker-like types, whereas a large number of
pacemaker-like cells was found during the differen-
tiation of β1-null ES cells, and only few atrial and
ventricular cells developed transiently (Fässler et al.,
1996). Also the development of sarcomeric proteins
was retarded in β1-null cardiomyocytes(Fässler et al.,
1996). The formation of myotubes was significantly
delayed and reduced, although some myocytes fused
and sarcomeres were formed. In the absence of β1in-
tegrin, ES cell-derived neurogenic differentiation was
accelerated, but the outgrowth of neuronal cells was
retarded (our unpublished data). We found an altered
expression pattern of regulatory genes involved in
mesodermal and neuroectodermal differentiation pos-
sibly due to an important regulatory function of β1
integrins during early cell lineage determination (our
unpublished data). It has been shown that the capacity
of the β1-null cells to differentiate into keratinocytes
in vitro was severely impaired since the differentiating
β1-null cells expressed the simple epithelial keratins,
but not K14, K10, and involucrin was detected only
occasionally (Bagutti et al., 1996).
In vitro differentiation of ES cells was also used
to demonstrate the disruption of visceral endoderm
differentiation after homozygous inactivation of the
GATA-4 (Soudais et al., 1995), and the inhibition of
myogenic differentiation after inactivation of desmin
genes(Weitzer et al., 1995).Inconclusion,in vitro dif-
ferentiation of mutant ES cells served as an excellent
alternative strategy in all those cases where the gene
defectresultedin early embryoniclethalityto unreveal
the function of those genes which are indispensable in
Summary and future prospects
TheES cell in vitro technologyallows thestudyofcel-
lular differentiation processes during early embryonic
developmentin culture. Thein vitro systems permitted
us to analyze the differentiation of early embryonic
cells via progenitorcells into highly differentiatedand
specialized cells of the cardiovascular, myogenic and
neurogenic lineages, as well as into epithelial and
VSM cells. From other laboratories, in vitro models
for hematopoietic (see Keller et al., 1995) and adi-
pogenic (Dani et al., 1997) differentiation of mouse
ES cells were successfully established.
During the last years, in addition to mice, other
organisms were successfully used to establish ES cell
lines, and living chimaeras have been obtained so far,
as for example, from chicken (Pain et al., 1996), fish
(Hong et al. 1996), rabbit (Schoonjans et al. 1996),
and pig (Wheeler, 1994).
These embryonic stem cell lines may be used to
investigate early developmental processes in vitro. In
addition, the effects of growth and differentiation fac-
tors (Wobus et al., 1994a, b; 1997b; Johansson and
Wiles, 1995) or extracellular matrix proteins on the
differentiationof embryonalcells may be investigated.
Furthermore, xenobiotics acting as embryotoxic and
teratogenic agents may be screened for their capacity
to modulate differentiation in vitro (Spielmann et al.,
1997). In addition, transgenic ES cell lines contain-
ing reporter genes fused to tissue-specific promoters
may be used to prescreen for embryotoxic/teratogenic
compounds which induce stage-specific developmen-
tal alterations in vitro. These embryotoxicological
approaches together with pharmacological and elec-
trophysiological analyses of differentiated ES cell-
derived cardiomyocytes (for analysis of cardioactive
drugs, see Wobus et al., 1991, 1994b, 1997b; Pich et
al., 1997) might help to reduce the use of laboratory
animals in pharmacotoxicology.
In addition,the differentiationof geneticallymodi-
fiedcells by“gainoffunction”(Rohwedeletal., 1995)
and “loss of function”(Fässler et al., 1996; Wobus and
Guan, 1998) using totipotent ES cells in vitro is an ex-
cellent alternative to and substitute for in vivo studies
with transgenic animals to analyse the consequences
of mutations during early embryogenesis. Inducible
gene trap experiments with ES cells will further help
to understand key events in early embryogenesis. By
using RA as differentiation inducer and transgenic ES
cell lines which express reporter genes, i.e. LacZ or
greenfluorescent protein(GFP; Cormacket al., 1996),
it is possible to screen selectively for genes which are
induced or repressed in a defined lineage (Forrester et
of special interest to obtain homogeneous populations
of differentiated cells to be used for transplantations.
For example, ES cells differentiated into GABAer-
gic (Strübing et al., 1995; Dinsmore et al., 1996) or
dopaminergic(Dinsmoreet al., 1998)neurons,cardiac
ventricular cells (Wobus et al., 1997b) or hematopoi-
etic cells (Hole and Smith, 1994; Potocnik et al. 1997)
might be used as a novel source of cells for somatic
therapy and transplantation. The construction of effi-
cient vector systems and selection strategies will open
further prospects for in vivo gene expression strate-
gies (Koh et al., 1995; Rust et al., 1997). Transgenic
ES cell lines carrying tissue-specific promoters fused
to selectable marker genes can be differentiated into
the specific lineages in vitro, and after selection in
vitro, the differentiated cell population might be used
in grafting experiments and transplantations to recon-
stitute defective tissues (Koh et al., 1995; Klug et al.,
1996; Dinsmore et al., 1996; Rust et al., 1997).
We wish to thank Mrs. S. Sommerfeld, K. Meier
and O. Weiß for skilful technical assistance, Drs. D.
Fürst, University of Potsdam, and R. Kemler, MPI for
Immunobiology, Freiburg, for providing monoclonal
antibodies, R. Fässler, University of Lund, for β1
integrin-null ES cells, and U. Pich for help with the
confocal scanning microscopy analysis. We are grate-
ful to all colleagues, especially to Dr. J. Hescheler,
University of Köln, for continuous collaboration. The
work was supported by the Deutsche Forschungsge-
meinschaft (Wo 503/1-3, SFB 366/YE1) and Fonds
der Chemischen Industrie (FCI).
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