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In vitro cellular models for cardiac development and pharmacotoxicology
Toxic.in VitroVol. 9. No. 4, pp. 477-488, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/95 $9.50 + 0.00
0887-2333(%)00023-2
Target Organs: Cardiac, Embryonic and Skin In Vitro Cellular Models for Cardiac
Development
and Pharmacotoxicology
A. M. WOBUS*, J. ROHWEDEL, Institute
V. MALTSEV and J. HESCHELERT
of Plant Genetics and Crop Plant Research, D-06466 Gatersleben and TInstitute of Pharmacology, FU Berlin, D-14195 Berlin, Germany
Abstract-Permanent cultures of cardiac cells described so far have limited value for studying cell biology and pharmacology of the developing heart because of the loss of proliferative capacity and cardiac-specific properties of cardiomyocytes during long-term cultivation. Pluripotent embryonic carcinoma (EC) and embryonic stem (ES) cells cultivated as permanent lines offer a new approach for studying cardiogenic differentiation in vitro. We describe cardiogenesis in vitro by differentiating EC and ES cells by way of embryo-like aggregates (embryoid bodies) into spontaneously beating cardiomyocytes. During cardiomyocyte differentiation three distinct developmental stages were defined by expression of specific action potentials and ionic currents measured by the whole-cell patchxlamp technique. Whereas early differentiated cardiomyocytes are characterized by action potentials and ionic currents typical for early pacemaker cells, terminally differentiated cardiomyocytes show action potentials and ionic currents inherent to ventricular-, atrial- or sinus nodal-like cells. These functional characteristics are in accordance with the expression of a- and B-cardiac myosin heavy chain at early differentiation stages and the additional expression of ventricular-specific MLC-2V and atrial-specific ANF genes at terminal stages demonstrated by reverse transcription polymerase chain reaction (RT-PCR) analysis. Pharmacological studies performed by measuring chronotropic responses and by analysing the Ca*+ channel activity correspond to data obtained with cardiac cells from living organisms. For testing the influence of exogenous compounds on cardiac differentiation the teratogenic compound retinoic acid (RA) was applied during distinct stages of embryoid body development. A temporally controlled influence of RA on cardiac differentiation and expression of cardiac-specific genes was found. We conclude that ES cell-derived cardiomyocytes provide an excellent cellular model to study early cardiac development and to perform pharmacological and embryotoxicological investigations.
Introduction During the last decades many approaches were established to study cardiac development in vertebrates, the differentiation of cardiac precursor cells into specialized cell types, as well as the development of physiological properties of cardiomyocytes during embryological development. Whereas much is known about development of the avian heart (Sperelakis,
1989; Sperelakis and Pappano, 1983), mammalian studies are hampered by rapid developmental changes during cardiac differentiation and specialization, the lack of molecular markers for stage-specific cardiomyocyte development and the lack of permanent cell lines that mimic various stages of cardiac growth and development (Litvin et al., 1992). *Author for correspondence at: Institut fiir Ptlanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-06466 Gatersleben; Germany. Abbreviations: DMSO = dimethvl sulfoxide: EC = embrvonic carcinoma; ES = embjonic stem;. RA = retin;c acid; RAR = RA receptor; RT-PCR = reverse transcription polymerase chain reaction.
The mammalian heart develops from the anterior lateral plate of splanchnic mesoderm. Presumptive heart cells migrate anteriorly between ectoderm and endoderm remaining in close contact with endodermal cells. This process of migration is influenced by complex cell-cell interactions and influences of growth and extracellular matrix factors. The two heart-forming primordia begin to fuse forming a single, primitive tubular heart. In mice, at 8.5 days post conceptionem (PC), the heart is developed into an atria1 chamber, a common ventricular chamber which is in direct continuity with the bulbus cordis region of the primitive heart, and this is in direct continuity with the outflow tract of the heart, which later on is involved in the formation of the sinus node. At this 8-10 somite developmental stage, the heart is the first functional and most prominent organ system of the 8.5day embryo (Kaufman, 1992). Although considerable data exist about skeletal 1992; Olson, 1990; myogenesis (Buckingham, Weintraub et al., 1991) limited knowledge is available about the origin, commitment and differentiation of cardiac cells forming the functioning heart in
A. M. Wobus et al.
478
mammals. Several data suggest that a very short time interval exists between mesodermal commitment and phenotypic manifestation of differentiated cardiomyocytes (Han et al., 1992). Recent developments in cell biology with transgenic, transfection and in vitro differentiation strategies should help to give more insight into processes regulating commitment and differentiation of cardiac structure (Chien et al.. 1993; Field, 1993; Litvin et al., 1992; Parker and Schneider, 1991; Robbins, 1993). The present paper reviews recent data of mammalian cardiomyocyte cultures with special emphasis on our results of in vitro cardiomyocyte differentiation from permanent lines of pluripotent ES cells. These studies, for the first time, give more insight into the cardiac specification during the very early embryonal development on a functional basis (Maltsev et al., 1993 and 1994; Wobus et al., 1991) and will also stimulate further attempts concerning the development of in vitro cellular systems in pharmacology and toxicology thus reducing the use of living animals in pharmacotoxicology.
drawn from the cell cycle. Therefore, many attempts were made to immortalize cardiogenic cells (Caviedes et al., 1993; Jaffredo et al., 1991; Sen et al., 1988) and establish cardiac cultures from transgenic cells (Guzman et al., 1993) or mammalian organisms (Delcarpio et al., 1991; Lanson et al., 1992; Steinhelper et al., 1990; see also Field, 1993). Some of the most frequently studied cardiac or cardiac-like lines are described in Table 1. Besides H9c2 cells cultivated from rat embryonic ventricle (Kimes and Brandt, 1976), transformed RCVC cells from adult rat ventricle (Caviedes et al., 1993) and immortalized AT-l cells established from right atria1 tumour of transgenic mice (Delcarpio et al., 1991; Steinhelper et al., 1990), cardiomyocytes can also be developed from permanent lines of mouse pluripotent undifferentiated EC and ES cells. It was found that cardiomyocytes developed from EC or ES cells are highly differentiated with respect to cardiac-specific gene expression, pharmacological and electrophysiological properties. For ES cells, they retain all functional properties characteristic for cardiac cells from mammalian organisms (Maltsev et al., 1993 and 1994; Wobus et al., 1991; see also Miller-Hance et al., 1993; Robbins et al., 1990).
In vitro systems of mammalian cardiac cells Different approaches have been developed for heart myocyte culture of mammalian organisms. Most of the earlier studies were performed on primary cultures of embryonal, neonatal or adult heart tissues (for example, see Bugaisky and Zak, 1989; Chevalier et al., 1987; Claycomb and Lanson, 1984; Isenberg and Klockner, 1982; Wollenberger, 1985). The use of permanent cell lines of myocardial tissue would have important advantages over primary cultures because of the availability of large amounts of a homogenous cell population at a definite developmental stage. Adult cardiomyocytes are generally viewed as terminally differentiated cells that have permanently with-
I.
Table Cell
hne
H9c2
Permanent
cardiac-like
ES cellderived cardiomyocytes recapitulate early cardiac development Pluripotent EC and totipotent ES cells established as permanent lines from teratocarcinomas or directly from undifferentiated cells of early embryos, respectively, retain in vitro the capacity to differentiate into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm (Evans and Kaufman, 1981; Martin, 1981; Martin and Evans, 1975). EC cells of line PI9 (McBurney and Rogers,
cell lines and ditTerentiated
cardiomyocytes
of mammalian
Rat embryonic
ventricle
Transient
origin
References
Characteristics
Origin
rectifying
K+
current,
mg sites, /i-adrenoceptors, and cardiac
isoform
dihydropyridine
bind-
G, and G, or-subunits,
skeletal
Hescheler Brand, al.
of z, Ca”Ch
et al. 1991; Kimes 1976;
1994;
and
Meija-Alvarez
Sipido
and
ef
Marban,
1991. RCVC
Transformed adult
cells of
1.Sarcomertc receptors,
rat ventricle
actin.
T-and
KC current,
a-actin.
L-type
desmin,
Ca’+
current,
no spontaneous
Caviedes
dihydropyridine delayed
electrical
et cl/. 1986 and
1993
rectifying
activity,
no Nat
channels AT-I
Immortahzed mouse
cells of
right
atrial
$-Cardiac
MHC.
s-cardiac
ANF.
desmin.
cardiac
Atrial
and ventricular
actin,
/r-actin,
connexin
43
Delcarplo
er al. 199 I
al. 1992; Steinheltxr
troponin,
: Lanson
el
et al. 1990.
turnour PI9
D3
Mouse
embryomc
carcinoma
cells (ECC),
‘embryoid
body’-
diac and skeletal intercalated
differentiation
ceptors,
+ induction
noceptors
Mouse
embryonic
cells (ESC),
stem
‘embryoid
body’differentation
= q-subunit
of L-type
Ca2+
channel
MLC-IA,
action
potentials: I,,,
atrial-,
IK.&,
= myosin
currents,
1,.
heavy
disks,
ventricular-. If, I,,.
chain
q-,
MLC
car-
Z-bands,
er al.
1990: Wobus
er al.
1994a.
ANF,
Doetschmann
er al. 1985; Malt-
choli-
MLCwith
Z-
cholinoceptors; sinus nodal-like;
I,,,,,,
Smith el al. 1987; Rudnicki
&adreno-
myofibrils
muscarinic
IN..
with
no muscarinic
MLC-IV,
intercalated
OL,-, p-adrenoceptors,
MLC-ZA,
myofibrils
Cd’+
bands,
MHC
MLC-IV,
cholinoceptors,
MHC,
qCa2+Ch,
currents: or,CaCh
discs, L-type
nicotinic
a-, D-Cardiac ZV,
MHC,
actin mRNA,
sev et al.
1993 and
bins et al.
1994; Rob-
1990; Sanchez
1991; Wobus
et al.
er al.
1991.
&wh
= myosin
light
chain
ANF
= atrial
natriuretic
factor
Model for cardiac development 1982) and ES cells of line D3 (Doetschmann
et al., 1985) and Bll7 (Wobus et al., 1991) were differentiated in culture by embryo-like aggregates, the ‘embryoid bodies’, into spontaneously beating cardiomyocytes. We developed a standardized cultivation system by differentiating a defined number of EC or ES cells in ‘hanging drops’ into embryoid bodies (Wobus et al., 1991 and 1994a). In contrast to ES cells, which already differentiate spontaneously, EC cells have to be treated during differentiation with chemical inducers, such as RA or dimethyl sulfoxide (DMSO) (Edwards et al., 1983; Jones-Villeneuve et al., 1982; McBurney et al., 1982; Wobus et al., 1994a). The ES cell-derived embryoid body aggregates were cultivated for 7 days in suspension and plated afterwards onto adhesive substrates. Cardiomyocytes develop further during cultivation and differentiation in the embryoid body outgrowth forming distinct clusters of spontaneously and synchronously pulsating cells within cells of other lineages, as for example, endodermal and epithelial cells, skeletal myocytes, as well as haematopoietic cells and angiogenic structures at terminal differentiation stages (Doetschmann et al., 1985; Maltsev et al., 1993; Miller-Hance et al., 1993; Risau et al., 1988; Wang et al., 1992; Wiles and Keller, 1991; Wobus et al., 1991). After embryoid body plating cardiomyocyte development was analysed for the estimation of different parameters by several methods: (i) measurements of chronotropic responses by adding cardioactive drugs to in vitro differentiated cardiomyocytes; (ii) analysis of cardiac-specific gene expression by the RT-PCR; (iii) identification of intracellular proteins by immunofluoresccnce with muscle- and cardiac-specific monoclonal antibodies; and (iv) analysis of functional properties, as for and ionic currents example, action potentials recorded by the patch-clamp technique (Hamill et al., 1981) on single isolated cardiomyocytes (Fig. 1). By characterizing the electrophysiological properties of cardiomyocytes with the patch-clamp technique, we found that during cardiomyogenesis in vitro three distinct developmental stages of cardiomyocyte differentiation could be determined: ‘early pacemaker’ cells (‘7 + 2 d’ to ‘7 + 4 d’), ‘intermediate stage’ cells (‘7 + 5 d’ to ‘7 + 8 d’) and cells at a ‘terminal differentiation stage’ (‘7 + 9 d’ to ‘7 + 18 d’), all characterized by a different and characteristic pattern of action potentials, ion channels and expression pattern of cardiac-specific genes (Fig. 2). Whereas early differentiated cardiomyocytes were characterized by action potentials of early pacemaker cells typical for embryonic cardiomyocytes, cardiomyocytes of terminal differentiation stage were characterized by specialized action potential patterns of atrial-, ventricular- and sinus nodal-like types, which were found to be hormonally and pharmacologically regulated (Maltsev et al., 1993). Corresponding to the action potential pattern the differentiated cardiomyocytes functionally expressed
479
the characteristic sets of ion channels of atrial, ventricular and sinus nodal cells of the heart (Fig. 2A). Whereas the first pulsating cardiomyocytes were observed in a few (2-5%) of the ‘7 d’ embryoid bodies, nearly all (95%) embryoid bodies measured in the time interval between ‘7 + 3 d’ to ‘7 + 10 d’ contained areas of pulsating cardiomyocytes. At the early differentiation stage, the most prominent ion current component characteristic for early pacemaker cells were voltage-dependent L-type Ca2+ channels which timedependently increased their density (Fig. 2C, see Maltsev et al., 1994). In parallel, the transcription of the a,-subunit of the Ca2+ channel gene (a, CaCh) was upregulated with differentiation time (Fig. 2B and C; unpublished data). The inward Na+ current (Bendorf et al., 1985), INa, was measured for the first time in cardiomyocytes of the intermediate stage, when the specialization into atrial-like cells occasionally takes place (Fig. 2C), but is increased in density during differentiation into the terminal differentiation stage. This is in agreement with the observation that the gene encoding the atrial natriuretic factor (ANF) is expressed at a very low level during this intermediate stage, but is expressed significantly at terminal differentiation stage (Fig. 2B) when cardiomyocytes of atrial-like type are clearly present (Fig. 2A). From the electrophysiological point of view expression of atrial- and sinus nodal-like cells are characterized by the inwardly rectifying muscarinic acetylcholineactivated K+ current, IK,ACh,known to be absent in ventricular-like cells (for comparison of pharmacological effects in EC and ES cells, see Fig. 3). Sinusnodal-like cells are characterized by a low expression of Na+ channels and inwardly rectifying K+ channels, but ionic current components regulated by cardiotropic hormones, for example the pacemaker I,, IK,Ach and I,, currents (Di Francesco, 1993; Maltsev et al., 1994). Our electrophysiological analysis demonstrates that only with this functional assay were we able to characterize precisely the early developmental stages of ES cell-derived cardiomyocytes (Maltsev et al., 1993 and 1994; for review see also Wobus et al., 1994~). Pharmacological responses of EC and ES celldiierentiated cardiomyocytes EC and ES cell-differentiated cardiomyocytes were investigated for their chronotropic responses by adding cardiotropic drugs during the differentiation period (Table 2; see Wobus et al., 1991 and 1994a). In both cellular systems and cardiomyocytes of all developmental stages positive chronotropic effects were induced by the /3-adrenoceptor agonists adrenaline and isoprenaline, the direct activator of the adenylyl cyclase, forskolin, the inhibitor of phosphodiesterases, isobutylmethylxanthine, the a,adrenoceptor agonist, phenylephrine, and the activator of the L-type Ca2+ channel, BayK 8644. j?,-Adrenoceptors are not expressed in ES cell-derived
A. M. Wobus et al.
480
0
Zygote
Teratocarcinoma/Teratoma
Blastocyst
._ g .s ge, &? 85 2 QU k P) ‘2 5 $ V
Cultivation of ESC and ECC as embryoid bodies in hanging drops and in suspension for 7 days +
4 Plating of embryoid bodies and differentiation into cardiomyocytes
1 A. Pharmacological
studies by chronotropic
B. Gene expression
analysis with RT-PCR
C. Immunofluorescence D. Electrophysiological patch-clamp technique
measurements technique
with muscle- and cardiac-specific
antibodies
studies on enzymatically isolated single cells: for analysing action potentials and ionic currents
Fig. 1. Relationship between early embryos, embryonic carcinoma cells (ECC) and embryonic stem cells (ESC) and their differentiation into cardiomyocytes via embryoid body development in vitro.
cardiomyocytes of early differentiation stage, because the &adrenoceptor agonist clenbuterol resulted in positive chronotropic effects only in cardiomyocytes of intermediate and terminal stage (A. M. Wobus et al., 1991 and 1994a, unpublished data). The chronotropic effects were abolished by the specific antagonists (Fig. 3). The L-type Ca2+ channel blockers nisoldipine, isradipine, gallopamil and diltiazem resulted in a negative chronotropic effect (Wobus et al., 1991 and 1994a). In contrast, negative chronotropic effects induced by the muscarinic cholinoceptor agonist, carbachol, were only measured in cardiomyocytes differentiated from ES cells, but not from EC cells (Fig. 3; Wobus et al., 1991 and 1994a). Furthermore, EC cell-derived cardiomyocytes showed a positive chronotropic effect on carbachol
which was blocked by D-tubocurarine, suggesting the involvement of nicotinic cholinoceptors. It may be assumed that EC cell-derived cardiomyocytes have adopted, at least in part, some characteristics of skeletal muscle cells similar to those described for H9c2 cells (see Kimes and Brandt, 1976). This different developmental and pharmacological behaviour of ES and EC cell-derived cardiac cells may be explained by the fact that EC cells have to be induced by exogenous factors, as, for example, RA or DMSO, to differentiate into cardiac-like cells. It was suggested (K. R. Chien, personal communication) that, for example, RA induces differentiation into ventricularlike cells, findings which are in agreement to our observation that ES cell-derived cardiomyocytes differentiated in the presence of RA during specific
481
Model for cardiac development
A Cardiac cell type
Early pacemaker
Days
6
Terminal differentiation stage
Intermediate stage
d to : 7+2 d to ; 7+4d 7d
7+9 d to 7+18 d
7+5 d to 7+8 d
I
--
ICa IK,to IK INa IKl IK,ATP IK,ACh
h
+60
Action potentials
--
Atrial-like cells
OmV
ICa IK,to IK INa -80
IKI IK.ATP
Ventricular-like cells I , :
on channels
8
If
IK,ACh
j , I
IK,to
7d
:
7+2 d
7+5 d
a
++
++
++
++
++
P
++
j 0 i I i I I :
++
++
++
++
++
++
++
++
+
+
++
++
I, I,
B
E
ICa IK,to IK
ICa
ICa
a,CaCh
+
ANF
-
5
7
IK,to IK INa
+2
+4
+6
Sinus nodal-like cells
7+18 d
7+9/12 d
+8
+I0
+I2
+I4
+I6
+18
days
Fig. 2. Development of action potentials (A), ionic currents (A, C) and expression of cardiac-specific genes (B) in ES cell-derived cardiomyocytes. Both atrial- and ventricular-like types of action potentials were elicited in current clamp mode by current pulse stimulation (204 pA, 3 ms; for details see Maltsev et al., 1993 and 1994). The differentiation of cardiomyocytes with specialized action potentials during early, intermediate and terminal differentiation stage is demonstrated with respect to action potentials and ion currents (A) and expression of cardiac-specific genes (B). Furthermore, the development of ES cell-derived cardiomyocytes (in %) and the increase in density of Ca*+ and Na+ current during differentiation is presented (C). Ion currents: I, : L-Type Ca2+ current, I,,O.. transient K+ current, I,,: inward Na+ current, I, : outwardly rectifying K+ current, I,, : inwardly rectifying K+ current, IitAch: muscarinic acetylcholineATP-modulated K+ current, I,: hyperpolarization-activated pacemaker activated K+ current, I,,,,: current. Genes: a, /I: a- and b-cardiac myosin heavy chain, a,CaCh: a,-subtype of the L-type Ca*+ channel, ANF: atria1 natriuretic factor, MLC-2V: myosin light chain-2 of ventricular cells.
482
et al.
A. M. Wobus
Carbachol
Adrenaline (p, Isoprenallne (p, Clenbuteiol (p2) Phenylephrine (al)
Atroplne
Chdinoceptor
Adenylyi
cyciase
Isradipine Nisoldipine Gallopam Dlltiazem
Bay K 8644
Adrenoceptor N
N
.
Forskolln
Bisoprolol (PI) Prazosin (al) ICI 118.551 (pz)
-
_
_
IBMX
(ESC/ECCI Fig. 3. Pharmacological responses of EC and ES cell-derived cardiomyocytes and signal transduction pathways involved in CaZ+ current regulation (IBMX-isobutylmethylxanthine). ( + ) and ( - ) means activating and inhibiting activity on Ca2+ channel, respectively.
developmental
stages (for details, see Wobus et al., 1994b) did not respond to the muscarinic cholinoceptor agonist, carbachol (A. M. Wobus, unpublished data). Our data demonstrate that cardiac-specific agonists and antagonists are involved in the pharmacological regulation of the Cal+ channel and P-adrenoceptormodulated signal transduction pathway of EC and ES cell-derived cardiomyocytes, but that the muscarinic cholinoceptor response is only expressed in ES cell-derived cardiomyocytes (Wobus et al.,
in
uitvo
During the development of cardiac cells and their specialization into distinct functional regions, complex inductive interactions of different embryonic tissues and influences of growth factors and extracellular matrix proteins are involved. Growth factors which were found to be important for the modulation of early mesodermal lineage, for development and differentiation of cardiac tissue and for the regulation of proliferation of cardiac cells include transforming
Table 2. Pharmacologxal effects of cardioactlve drugs by measuring chronotropic effects at maximal etTwtive concentrations on EC cell (Pl9)- and ES cell (D3. BII7)-derived spontaneously beating cardiomyocytes summarized for ‘7 + 2 d’ measurements (+ =positive chronotropic response, - = negative chronotropic response; see Wobus PI al. 1991 and 1994a) Chronotropic (‘7+2 Substance (maximal concentration, PM)
effective
( - )Isoprenaline (IO) Isobutylmethylxanthine (10) Forskolin (I) Clenbuterol (I) Clenbuterof (I) (‘7 + 9 d’) ( - )Phenylephrine Carbachol (100) Isradipine (I) Nisoldipine (0. I) Gallopamil (I) Diltiazem (10) BayK 8644 (I)
Function fi-Adrenoceptor agonist Inhibitor of phosphodiesterase Activator of adenylyl cyclase /3-Adrenoceptor agonist P-Adrenoceptor agonist jT,-Adrenoceptor agonist Muscarinic cholinoceptor agonist Ca*+ channel blocker Ca*+ channel blocker Ca*’ channel blocker Ca*+ channel blocker Ca2+ channel opener ND = not determined
ES cell-derived cardiomyocytes + + + _
response d’) EC cell-derived cardiomyocytes + ND + + +
+ + _ _ -
(1) _ _ _ _
+
+
Embryoid
body
Plating
differentiation
5
-
(7 d)
Cardiomyocyti development [7 + 3 d]
‘ld
Plate. 1. Morphology of D3 ES cell-derived embryoid bodies differentiated for 2 (A), 5 (B) and 7 days (C) visualized by scanning electron microscopy and experimental scheme for studying the stage-specific effects of RA treatment on cardiomyocyte differentiation. Development of cardiomyocytes (or myocytes) was determined morphoiogicahy by immunofluorescence and RT-PCR analysis (a- and p-cardiac myosin heavy chain for cardiac-specific and myogenin for skeletal muscle cell-specific gene expression, see Wobus e/ al. 1994b). Bar = 50pm.
483
e
Model for cardiac development growth growth
factor-/3 (TGF-/I), factors (aFGF,
acidic and basic fibroblast
bFGF) and insulin-like growth factors (IGF-I and IGF-II; see Akhurst et al., 1990; Consigli and Joseph-Silverstein, 1991; Foster er al., 1989; Kardami and Fandrich, 1989; Kimelman and Kirschner, 1987; Parker and Schneider, 1991; Runyan et al., 1990). In addition, proteins of the extracellular matrix, as fibronectin, vitronectin, collagen and laminin are involved in cell-cell interactions, adhesion and migratory processes during the embryonal development of the functional heart (i.e. Borg et al., 1990; Linask and Lash, 1988; Price et al., 1992; Sinning et al., 1988; Sumida et al., 1990). The ES cell-derived embryoid body differentiation system has been used to analyse the effects of differentiation factors on embryonic development in vitro (Johansson and Wiles, 1994; van den Eijnden van Raaij et al., 1991; Wiles and Keller, 1991; Wobus et al., 1994b). To study the effects of exogenous factors on differentiation processes in vitro it is necessary to remove growth and differentiation factors normally present in the foetal calf serum of cultivation medium. Therefore, ES cells were differentiated by cultivating them in chemically defined media (Johansson and Wiles, 1995) or medium supplemented with growth factor-depleted serum (van den Eijnden van Raaij et al., 1991; Wobus et al., 1994b). We investigated the effect of the vitamin A analogue, RA, on the differentiation pattern of ES cells (Wobus et al., 1994b). RA is one of the most potent teratogens (Kochhar, 1973; Lammer et al., 1985), but is also known to function as morphogen (Eichele, 1989; Tabin, 1991), activator of Hoxgenes in mammals (Kessel and Gruss, 1991) and potent differentiation-inducing agent for in vitro cultivated cells (Jones-Villeneuve et al., 1982; Simeone et al., 1990). ES cells of line D3 were cultivated in the presence of RA during defined time intervals of embryoid body differentiation, from day 0 to 2, 2 to 5 and 5 to 7 (Fig. 4). These embryoid bodies reflect precise developmental stages, comparable with early embryos. For example, a 5-day embryoid body contains undifferentiated cells in the centre surrounded by endodermal cells, thus resembling a blastocyst stage embryo at the time of implantation. The 7- to 8-day embryoid body contains, in addition to endodermal and ectodermal derivatives, cells of the early mesoderm (Robertson, 1987). Developmental changes in the embryoid body morphology during differentiation can be seen clearly in the scanning electron microscope. Whereas ‘2 d’ embryoid bodies contained aggregates of nearly uncommitted ES cells, in the ‘5 d’ and even more in the ‘7 d’ embryoid bodies flat endodermal cells are developed on the surface of the embryoid body (Fig. 4AC). The ‘7 d’ embryoid bodies (treated with RA during different times) were plated and the formation of differentiated cell types was evaluated during further
485
cardiomyocyte differentiation (Fig. 4; Wobus et al., 1994b). ES cells treated during the first 2 days of embryoid body differentiation (‘0 to 2 d’) with high concentrations of RA resulted in a strong inhibition of cardiogenesis. The same concentrations of RA, when added between day 5 and 7 of embryoid body development (‘5 to 7 d’), however, induced no inhibition, but, in contrast, an induction of cardiomyocyte differentiation. ES cell-derived embryoid bodies cultivated in the presence of high RA concentrations between day 2 and 5 differentiated into skeletal myocytes instead into cardiomyocytes. This was demonstrated morphologically by immunofluorescence and RT-PCR analysis by time- and concentration-dependent inhibition of transcription of cardiac-specific myosin heavy chain (MHC) genes and the induction of transcription of skeletal musclespecific myogenin (Fig. 4). Furthermore, using the whole-ceil patch-clamp technique, we could demonstrate the functional activity of the induced myocytes by expression of skeletal muscle-specific CaZ+ channels and nicotinic cholinoceptor-operated channels. In summary, we may conclude that RA treatment during ectodermal (or neuroectodermal) differentiation (‘2 to 5 d’) resulted in a suppression of genes responsible for cardiomyocyte differentiation and in activation of genes involved in myogenesis. This stage-specific effect would imply that RA receptors (RARs) are present on embryoid bodies at these specific stages. In mice, the RAR subtypes a, /?and y were found to be specifically expressed in presomitic stage embryos in the lateral regions, during crest cell migration, and in the primitive streak region throughout the period of neurulation, respectively (Ruberte et al., 1991). Differentiating teratocarcinema cells showed significant alterations in the expression pattern of the RAR a, j and y subtypes especially during neuroectodermal differentiation: a strong up-regulation of RAR a and /I, and a rapid
down-regulation of RAR y mRNA was found suggesting a role for RAR a and fl expression during neuroectodermal differentiation (Jonk et al., 1992; Kruyt et al., 1991). As ES cells differentiating in embryoid bodies apparently reflect the specific time- and concentration-dependent effects of RA on embryonic development this ES cell-differentiation system provides a new model to study embryonic development under defined conditions in vitro and investigate the influence of exogenous factors during early embryogenesis for in vitro embryotoxicological studies.
Acknowlednemenrs-This
research was suDnorted by the
Deutsche sorsehungsgemeinschaft, the M&i&y of Science and Research of Sachsen-Anhalt. ZEBET. Robert-KochInstitute of Germany, Fonds der dhemischen Industrie and BAYER, Wuppertal, Gennany. We thank Dr Klaus Adler, IPK Gatersleben, for scanning electron microscopy of embryoid bodies and Dr Michael Wiles, Base1 Institute of Immunology, for submitting unpublished data.
486
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0887-2333(%)00023-2
Target Organs: Cardiac, Embryonic and Skin In Vitro Cellular Models for Cardiac
Development
and Pharmacotoxicology
A. M. WOBUS*, J. ROHWEDEL, Institute
V. MALTSEV and J. HESCHELERT
of Plant Genetics and Crop Plant Research, D-06466 Gatersleben and TInstitute of Pharmacology, FU Berlin, D-14195 Berlin, Germany
Abstract-Permanent cultures of cardiac cells described so far have limited value for studying cell biology and pharmacology of the developing heart because of the loss of proliferative capacity and cardiac-specific properties of cardiomyocytes during long-term cultivation. Pluripotent embryonic carcinoma (EC) and embryonic stem (ES) cells cultivated as permanent lines offer a new approach for studying cardiogenic differentiation in vitro. We describe cardiogenesis in vitro by differentiating EC and ES cells by way of embryo-like aggregates (embryoid bodies) into spontaneously beating cardiomyocytes. During cardiomyocyte differentiation three distinct developmental stages were defined by expression of specific action potentials and ionic currents measured by the whole-cell patchxlamp technique. Whereas early differentiated cardiomyocytes are characterized by action potentials and ionic currents typical for early pacemaker cells, terminally differentiated cardiomyocytes show action potentials and ionic currents inherent to ventricular-, atrial- or sinus nodal-like cells. These functional characteristics are in accordance with the expression of a- and B-cardiac myosin heavy chain at early differentiation stages and the additional expression of ventricular-specific MLC-2V and atrial-specific ANF genes at terminal stages demonstrated by reverse transcription polymerase chain reaction (RT-PCR) analysis. Pharmacological studies performed by measuring chronotropic responses and by analysing the Ca*+ channel activity correspond to data obtained with cardiac cells from living organisms. For testing the influence of exogenous compounds on cardiac differentiation the teratogenic compound retinoic acid (RA) was applied during distinct stages of embryoid body development. A temporally controlled influence of RA on cardiac differentiation and expression of cardiac-specific genes was found. We conclude that ES cell-derived cardiomyocytes provide an excellent cellular model to study early cardiac development and to perform pharmacological and embryotoxicological investigations.
Introduction During the last decades many approaches were established to study cardiac development in vertebrates, the differentiation of cardiac precursor cells into specialized cell types, as well as the development of physiological properties of cardiomyocytes during embryological development. Whereas much is known about development of the avian heart (Sperelakis,
1989; Sperelakis and Pappano, 1983), mammalian studies are hampered by rapid developmental changes during cardiac differentiation and specialization, the lack of molecular markers for stage-specific cardiomyocyte development and the lack of permanent cell lines that mimic various stages of cardiac growth and development (Litvin et al., 1992). *Author for correspondence at: Institut fiir Ptlanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-06466 Gatersleben; Germany. Abbreviations: DMSO = dimethvl sulfoxide: EC = embrvonic carcinoma; ES = embjonic stem;. RA = retin;c acid; RAR = RA receptor; RT-PCR = reverse transcription polymerase chain reaction.
The mammalian heart develops from the anterior lateral plate of splanchnic mesoderm. Presumptive heart cells migrate anteriorly between ectoderm and endoderm remaining in close contact with endodermal cells. This process of migration is influenced by complex cell-cell interactions and influences of growth and extracellular matrix factors. The two heart-forming primordia begin to fuse forming a single, primitive tubular heart. In mice, at 8.5 days post conceptionem (PC), the heart is developed into an atria1 chamber, a common ventricular chamber which is in direct continuity with the bulbus cordis region of the primitive heart, and this is in direct continuity with the outflow tract of the heart, which later on is involved in the formation of the sinus node. At this 8-10 somite developmental stage, the heart is the first functional and most prominent organ system of the 8.5day embryo (Kaufman, 1992). Although considerable data exist about skeletal 1992; Olson, 1990; myogenesis (Buckingham, Weintraub et al., 1991) limited knowledge is available about the origin, commitment and differentiation of cardiac cells forming the functioning heart in
A. M. Wobus et al.
478
mammals. Several data suggest that a very short time interval exists between mesodermal commitment and phenotypic manifestation of differentiated cardiomyocytes (Han et al., 1992). Recent developments in cell biology with transgenic, transfection and in vitro differentiation strategies should help to give more insight into processes regulating commitment and differentiation of cardiac structure (Chien et al.. 1993; Field, 1993; Litvin et al., 1992; Parker and Schneider, 1991; Robbins, 1993). The present paper reviews recent data of mammalian cardiomyocyte cultures with special emphasis on our results of in vitro cardiomyocyte differentiation from permanent lines of pluripotent ES cells. These studies, for the first time, give more insight into the cardiac specification during the very early embryonal development on a functional basis (Maltsev et al., 1993 and 1994; Wobus et al., 1991) and will also stimulate further attempts concerning the development of in vitro cellular systems in pharmacology and toxicology thus reducing the use of living animals in pharmacotoxicology.
drawn from the cell cycle. Therefore, many attempts were made to immortalize cardiogenic cells (Caviedes et al., 1993; Jaffredo et al., 1991; Sen et al., 1988) and establish cardiac cultures from transgenic cells (Guzman et al., 1993) or mammalian organisms (Delcarpio et al., 1991; Lanson et al., 1992; Steinhelper et al., 1990; see also Field, 1993). Some of the most frequently studied cardiac or cardiac-like lines are described in Table 1. Besides H9c2 cells cultivated from rat embryonic ventricle (Kimes and Brandt, 1976), transformed RCVC cells from adult rat ventricle (Caviedes et al., 1993) and immortalized AT-l cells established from right atria1 tumour of transgenic mice (Delcarpio et al., 1991; Steinhelper et al., 1990), cardiomyocytes can also be developed from permanent lines of mouse pluripotent undifferentiated EC and ES cells. It was found that cardiomyocytes developed from EC or ES cells are highly differentiated with respect to cardiac-specific gene expression, pharmacological and electrophysiological properties. For ES cells, they retain all functional properties characteristic for cardiac cells from mammalian organisms (Maltsev et al., 1993 and 1994; Wobus et al., 1991; see also Miller-Hance et al., 1993; Robbins et al., 1990).
In vitro systems of mammalian cardiac cells Different approaches have been developed for heart myocyte culture of mammalian organisms. Most of the earlier studies were performed on primary cultures of embryonal, neonatal or adult heart tissues (for example, see Bugaisky and Zak, 1989; Chevalier et al., 1987; Claycomb and Lanson, 1984; Isenberg and Klockner, 1982; Wollenberger, 1985). The use of permanent cell lines of myocardial tissue would have important advantages over primary cultures because of the availability of large amounts of a homogenous cell population at a definite developmental stage. Adult cardiomyocytes are generally viewed as terminally differentiated cells that have permanently with-
I.
Table Cell
hne
H9c2
Permanent
cardiac-like
ES cellderived cardiomyocytes recapitulate early cardiac development Pluripotent EC and totipotent ES cells established as permanent lines from teratocarcinomas or directly from undifferentiated cells of early embryos, respectively, retain in vitro the capacity to differentiate into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm (Evans and Kaufman, 1981; Martin, 1981; Martin and Evans, 1975). EC cells of line PI9 (McBurney and Rogers,
cell lines and ditTerentiated
cardiomyocytes
of mammalian
Rat embryonic
ventricle
Transient
origin
References
Characteristics
Origin
rectifying
K+
current,
mg sites, /i-adrenoceptors, and cardiac
isoform
dihydropyridine
bind-
G, and G, or-subunits,
skeletal
Hescheler Brand, al.
of z, Ca”Ch
et al. 1991; Kimes 1976;
1994;
and
Meija-Alvarez
Sipido
and
ef
Marban,
1991. RCVC
Transformed adult
cells of
1.Sarcomertc receptors,
rat ventricle
actin.
T-and
KC current,
a-actin.
L-type
desmin,
Ca’+
current,
no spontaneous
Caviedes
dihydropyridine delayed
electrical
et cl/. 1986 and
1993
rectifying
activity,
no Nat
channels AT-I
Immortahzed mouse
cells of
right
atrial
$-Cardiac
MHC.
s-cardiac
ANF.
desmin.
cardiac
Atrial
and ventricular
actin,
/r-actin,
connexin
43
Delcarplo
er al. 199 I
al. 1992; Steinheltxr
troponin,
: Lanson
el
et al. 1990.
turnour PI9
D3
Mouse
embryomc
carcinoma
cells (ECC),
‘embryoid
body’-
diac and skeletal intercalated
differentiation
ceptors,
+ induction
noceptors
Mouse
embryonic
cells (ESC),
stem
‘embryoid
body’differentation
= q-subunit
of L-type
Ca2+
channel
MLC-IA,
action
potentials: I,,,
atrial-,
IK.&,
= myosin
currents,
1,.
heavy
disks,
ventricular-. If, I,,.
chain
q-,
MLC
car-
Z-bands,
er al.
1990: Wobus
er al.
1994a.
ANF,
Doetschmann
er al. 1985; Malt-
choli-
MLCwith
Z-
cholinoceptors; sinus nodal-like;
I,,,,,,
Smith el al. 1987; Rudnicki
&adreno-
myofibrils
muscarinic
IN..
with
no muscarinic
MLC-IV,
intercalated
OL,-, p-adrenoceptors,
MLC-ZA,
myofibrils
Cd’+
bands,
MHC
MLC-IV,
cholinoceptors,
MHC,
qCa2+Ch,
currents: or,CaCh
discs, L-type
nicotinic
a-, D-Cardiac ZV,
MHC,
actin mRNA,
sev et al.
1993 and
bins et al.
1994; Rob-
1990; Sanchez
1991; Wobus
et al.
er al.
1991.
&wh
= myosin
light
chain
ANF
= atrial
natriuretic
factor
Model for cardiac development 1982) and ES cells of line D3 (Doetschmann
et al., 1985) and Bll7 (Wobus et al., 1991) were differentiated in culture by embryo-like aggregates, the ‘embryoid bodies’, into spontaneously beating cardiomyocytes. We developed a standardized cultivation system by differentiating a defined number of EC or ES cells in ‘hanging drops’ into embryoid bodies (Wobus et al., 1991 and 1994a). In contrast to ES cells, which already differentiate spontaneously, EC cells have to be treated during differentiation with chemical inducers, such as RA or dimethyl sulfoxide (DMSO) (Edwards et al., 1983; Jones-Villeneuve et al., 1982; McBurney et al., 1982; Wobus et al., 1994a). The ES cell-derived embryoid body aggregates were cultivated for 7 days in suspension and plated afterwards onto adhesive substrates. Cardiomyocytes develop further during cultivation and differentiation in the embryoid body outgrowth forming distinct clusters of spontaneously and synchronously pulsating cells within cells of other lineages, as for example, endodermal and epithelial cells, skeletal myocytes, as well as haematopoietic cells and angiogenic structures at terminal differentiation stages (Doetschmann et al., 1985; Maltsev et al., 1993; Miller-Hance et al., 1993; Risau et al., 1988; Wang et al., 1992; Wiles and Keller, 1991; Wobus et al., 1991). After embryoid body plating cardiomyocyte development was analysed for the estimation of different parameters by several methods: (i) measurements of chronotropic responses by adding cardioactive drugs to in vitro differentiated cardiomyocytes; (ii) analysis of cardiac-specific gene expression by the RT-PCR; (iii) identification of intracellular proteins by immunofluoresccnce with muscle- and cardiac-specific monoclonal antibodies; and (iv) analysis of functional properties, as for and ionic currents example, action potentials recorded by the patch-clamp technique (Hamill et al., 1981) on single isolated cardiomyocytes (Fig. 1). By characterizing the electrophysiological properties of cardiomyocytes with the patch-clamp technique, we found that during cardiomyogenesis in vitro three distinct developmental stages of cardiomyocyte differentiation could be determined: ‘early pacemaker’ cells (‘7 + 2 d’ to ‘7 + 4 d’), ‘intermediate stage’ cells (‘7 + 5 d’ to ‘7 + 8 d’) and cells at a ‘terminal differentiation stage’ (‘7 + 9 d’ to ‘7 + 18 d’), all characterized by a different and characteristic pattern of action potentials, ion channels and expression pattern of cardiac-specific genes (Fig. 2). Whereas early differentiated cardiomyocytes were characterized by action potentials of early pacemaker cells typical for embryonic cardiomyocytes, cardiomyocytes of terminal differentiation stage were characterized by specialized action potential patterns of atrial-, ventricular- and sinus nodal-like types, which were found to be hormonally and pharmacologically regulated (Maltsev et al., 1993). Corresponding to the action potential pattern the differentiated cardiomyocytes functionally expressed
479
the characteristic sets of ion channels of atrial, ventricular and sinus nodal cells of the heart (Fig. 2A). Whereas the first pulsating cardiomyocytes were observed in a few (2-5%) of the ‘7 d’ embryoid bodies, nearly all (95%) embryoid bodies measured in the time interval between ‘7 + 3 d’ to ‘7 + 10 d’ contained areas of pulsating cardiomyocytes. At the early differentiation stage, the most prominent ion current component characteristic for early pacemaker cells were voltage-dependent L-type Ca2+ channels which timedependently increased their density (Fig. 2C, see Maltsev et al., 1994). In parallel, the transcription of the a,-subunit of the Ca2+ channel gene (a, CaCh) was upregulated with differentiation time (Fig. 2B and C; unpublished data). The inward Na+ current (Bendorf et al., 1985), INa, was measured for the first time in cardiomyocytes of the intermediate stage, when the specialization into atrial-like cells occasionally takes place (Fig. 2C), but is increased in density during differentiation into the terminal differentiation stage. This is in agreement with the observation that the gene encoding the atrial natriuretic factor (ANF) is expressed at a very low level during this intermediate stage, but is expressed significantly at terminal differentiation stage (Fig. 2B) when cardiomyocytes of atrial-like type are clearly present (Fig. 2A). From the electrophysiological point of view expression of atrial- and sinus nodal-like cells are characterized by the inwardly rectifying muscarinic acetylcholineactivated K+ current, IK,ACh,known to be absent in ventricular-like cells (for comparison of pharmacological effects in EC and ES cells, see Fig. 3). Sinusnodal-like cells are characterized by a low expression of Na+ channels and inwardly rectifying K+ channels, but ionic current components regulated by cardiotropic hormones, for example the pacemaker I,, IK,Ach and I,, currents (Di Francesco, 1993; Maltsev et al., 1994). Our electrophysiological analysis demonstrates that only with this functional assay were we able to characterize precisely the early developmental stages of ES cell-derived cardiomyocytes (Maltsev et al., 1993 and 1994; for review see also Wobus et al., 1994~). Pharmacological responses of EC and ES celldiierentiated cardiomyocytes EC and ES cell-differentiated cardiomyocytes were investigated for their chronotropic responses by adding cardiotropic drugs during the differentiation period (Table 2; see Wobus et al., 1991 and 1994a). In both cellular systems and cardiomyocytes of all developmental stages positive chronotropic effects were induced by the /3-adrenoceptor agonists adrenaline and isoprenaline, the direct activator of the adenylyl cyclase, forskolin, the inhibitor of phosphodiesterases, isobutylmethylxanthine, the a,adrenoceptor agonist, phenylephrine, and the activator of the L-type Ca2+ channel, BayK 8644. j?,-Adrenoceptors are not expressed in ES cell-derived
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0
Zygote
Teratocarcinoma/Teratoma
Blastocyst
._ g .s ge, &? 85 2 QU k P) ‘2 5 $ V
Cultivation of ESC and ECC as embryoid bodies in hanging drops and in suspension for 7 days +
4 Plating of embryoid bodies and differentiation into cardiomyocytes
1 A. Pharmacological
studies by chronotropic
B. Gene expression
analysis with RT-PCR
C. Immunofluorescence D. Electrophysiological patch-clamp technique
measurements technique
with muscle- and cardiac-specific
antibodies
studies on enzymatically isolated single cells: for analysing action potentials and ionic currents
Fig. 1. Relationship between early embryos, embryonic carcinoma cells (ECC) and embryonic stem cells (ESC) and their differentiation into cardiomyocytes via embryoid body development in vitro.
cardiomyocytes of early differentiation stage, because the &adrenoceptor agonist clenbuterol resulted in positive chronotropic effects only in cardiomyocytes of intermediate and terminal stage (A. M. Wobus et al., 1991 and 1994a, unpublished data). The chronotropic effects were abolished by the specific antagonists (Fig. 3). The L-type Ca2+ channel blockers nisoldipine, isradipine, gallopamil and diltiazem resulted in a negative chronotropic effect (Wobus et al., 1991 and 1994a). In contrast, negative chronotropic effects induced by the muscarinic cholinoceptor agonist, carbachol, were only measured in cardiomyocytes differentiated from ES cells, but not from EC cells (Fig. 3; Wobus et al., 1991 and 1994a). Furthermore, EC cell-derived cardiomyocytes showed a positive chronotropic effect on carbachol
which was blocked by D-tubocurarine, suggesting the involvement of nicotinic cholinoceptors. It may be assumed that EC cell-derived cardiomyocytes have adopted, at least in part, some characteristics of skeletal muscle cells similar to those described for H9c2 cells (see Kimes and Brandt, 1976). This different developmental and pharmacological behaviour of ES and EC cell-derived cardiac cells may be explained by the fact that EC cells have to be induced by exogenous factors, as, for example, RA or DMSO, to differentiate into cardiac-like cells. It was suggested (K. R. Chien, personal communication) that, for example, RA induces differentiation into ventricularlike cells, findings which are in agreement to our observation that ES cell-derived cardiomyocytes differentiated in the presence of RA during specific
481
Model for cardiac development
A Cardiac cell type
Early pacemaker
Days
6
Terminal differentiation stage
Intermediate stage
d to : 7+2 d to ; 7+4d 7d
7+9 d to 7+18 d
7+5 d to 7+8 d
I
--
ICa IK,to IK INa IKl IK,ATP IK,ACh
h
+60
Action potentials
--
Atrial-like cells
OmV
ICa IK,to IK INa -80
IKI IK.ATP
Ventricular-like cells I , :
on channels
8
If
IK,ACh
j , I
IK,to
7d
:
7+2 d
7+5 d
a
++
++
++
++
++
P
++
j 0 i I i I I :
++
++
++
++
++
++
++
++
+
+
++
++
I, I,
B
E
ICa IK,to IK
ICa
ICa
a,CaCh
+
ANF
-
5
7
IK,to IK INa
+2
+4
+6
Sinus nodal-like cells
7+18 d
7+9/12 d
+8
+I0
+I2
+I4
+I6
+18
days
Fig. 2. Development of action potentials (A), ionic currents (A, C) and expression of cardiac-specific genes (B) in ES cell-derived cardiomyocytes. Both atrial- and ventricular-like types of action potentials were elicited in current clamp mode by current pulse stimulation (204 pA, 3 ms; for details see Maltsev et al., 1993 and 1994). The differentiation of cardiomyocytes with specialized action potentials during early, intermediate and terminal differentiation stage is demonstrated with respect to action potentials and ion currents (A) and expression of cardiac-specific genes (B). Furthermore, the development of ES cell-derived cardiomyocytes (in %) and the increase in density of Ca*+ and Na+ current during differentiation is presented (C). Ion currents: I, : L-Type Ca2+ current, I,,O.. transient K+ current, I,,: inward Na+ current, I, : outwardly rectifying K+ current, I,, : inwardly rectifying K+ current, IitAch: muscarinic acetylcholineATP-modulated K+ current, I,: hyperpolarization-activated pacemaker activated K+ current, I,,,,: current. Genes: a, /I: a- and b-cardiac myosin heavy chain, a,CaCh: a,-subtype of the L-type Ca*+ channel, ANF: atria1 natriuretic factor, MLC-2V: myosin light chain-2 of ventricular cells.
482
et al.
A. M. Wobus
Carbachol
Adrenaline (p, Isoprenallne (p, Clenbuteiol (p2) Phenylephrine (al)
Atroplne
Chdinoceptor
Adenylyi
cyciase
Isradipine Nisoldipine Gallopam Dlltiazem
Bay K 8644
Adrenoceptor N
N
.
Forskolln
Bisoprolol (PI) Prazosin (al) ICI 118.551 (pz)
-
_
_
IBMX
(ESC/ECCI Fig. 3. Pharmacological responses of EC and ES cell-derived cardiomyocytes and signal transduction pathways involved in CaZ+ current regulation (IBMX-isobutylmethylxanthine). ( + ) and ( - ) means activating and inhibiting activity on Ca2+ channel, respectively.
developmental
stages (for details, see Wobus et al., 1994b) did not respond to the muscarinic cholinoceptor agonist, carbachol (A. M. Wobus, unpublished data). Our data demonstrate that cardiac-specific agonists and antagonists are involved in the pharmacological regulation of the Cal+ channel and P-adrenoceptormodulated signal transduction pathway of EC and ES cell-derived cardiomyocytes, but that the muscarinic cholinoceptor response is only expressed in ES cell-derived cardiomyocytes (Wobus et al.,
in
uitvo
During the development of cardiac cells and their specialization into distinct functional regions, complex inductive interactions of different embryonic tissues and influences of growth factors and extracellular matrix proteins are involved. Growth factors which were found to be important for the modulation of early mesodermal lineage, for development and differentiation of cardiac tissue and for the regulation of proliferation of cardiac cells include transforming
Table 2. Pharmacologxal effects of cardioactlve drugs by measuring chronotropic effects at maximal etTwtive concentrations on EC cell (Pl9)- and ES cell (D3. BII7)-derived spontaneously beating cardiomyocytes summarized for ‘7 + 2 d’ measurements (+ =positive chronotropic response, - = negative chronotropic response; see Wobus PI al. 1991 and 1994a) Chronotropic (‘7+2 Substance (maximal concentration, PM)
effective
( - )Isoprenaline (IO) Isobutylmethylxanthine (10) Forskolin (I) Clenbuterol (I) Clenbuterof (I) (‘7 + 9 d’) ( - )Phenylephrine Carbachol (100) Isradipine (I) Nisoldipine (0. I) Gallopamil (I) Diltiazem (10) BayK 8644 (I)
Function fi-Adrenoceptor agonist Inhibitor of phosphodiesterase Activator of adenylyl cyclase /3-Adrenoceptor agonist P-Adrenoceptor agonist jT,-Adrenoceptor agonist Muscarinic cholinoceptor agonist Ca*+ channel blocker Ca*+ channel blocker Ca*’ channel blocker Ca*+ channel blocker Ca2+ channel opener ND = not determined
ES cell-derived cardiomyocytes + + + _
response d’) EC cell-derived cardiomyocytes + ND + + +
+ + _ _ -
(1) _ _ _ _
+
+
Embryoid
body
Plating
differentiation
5
-
(7 d)
Cardiomyocyti development [7 + 3 d]
‘ld
Plate. 1. Morphology of D3 ES cell-derived embryoid bodies differentiated for 2 (A), 5 (B) and 7 days (C) visualized by scanning electron microscopy and experimental scheme for studying the stage-specific effects of RA treatment on cardiomyocyte differentiation. Development of cardiomyocytes (or myocytes) was determined morphoiogicahy by immunofluorescence and RT-PCR analysis (a- and p-cardiac myosin heavy chain for cardiac-specific and myogenin for skeletal muscle cell-specific gene expression, see Wobus e/ al. 1994b). Bar = 50pm.
483
e
Model for cardiac development growth growth
factor-/3 (TGF-/I), factors (aFGF,
acidic and basic fibroblast
bFGF) and insulin-like growth factors (IGF-I and IGF-II; see Akhurst et al., 1990; Consigli and Joseph-Silverstein, 1991; Foster er al., 1989; Kardami and Fandrich, 1989; Kimelman and Kirschner, 1987; Parker and Schneider, 1991; Runyan et al., 1990). In addition, proteins of the extracellular matrix, as fibronectin, vitronectin, collagen and laminin are involved in cell-cell interactions, adhesion and migratory processes during the embryonal development of the functional heart (i.e. Borg et al., 1990; Linask and Lash, 1988; Price et al., 1992; Sinning et al., 1988; Sumida et al., 1990). The ES cell-derived embryoid body differentiation system has been used to analyse the effects of differentiation factors on embryonic development in vitro (Johansson and Wiles, 1994; van den Eijnden van Raaij et al., 1991; Wiles and Keller, 1991; Wobus et al., 1994b). To study the effects of exogenous factors on differentiation processes in vitro it is necessary to remove growth and differentiation factors normally present in the foetal calf serum of cultivation medium. Therefore, ES cells were differentiated by cultivating them in chemically defined media (Johansson and Wiles, 1995) or medium supplemented with growth factor-depleted serum (van den Eijnden van Raaij et al., 1991; Wobus et al., 1994b). We investigated the effect of the vitamin A analogue, RA, on the differentiation pattern of ES cells (Wobus et al., 1994b). RA is one of the most potent teratogens (Kochhar, 1973; Lammer et al., 1985), but is also known to function as morphogen (Eichele, 1989; Tabin, 1991), activator of Hoxgenes in mammals (Kessel and Gruss, 1991) and potent differentiation-inducing agent for in vitro cultivated cells (Jones-Villeneuve et al., 1982; Simeone et al., 1990). ES cells of line D3 were cultivated in the presence of RA during defined time intervals of embryoid body differentiation, from day 0 to 2, 2 to 5 and 5 to 7 (Fig. 4). These embryoid bodies reflect precise developmental stages, comparable with early embryos. For example, a 5-day embryoid body contains undifferentiated cells in the centre surrounded by endodermal cells, thus resembling a blastocyst stage embryo at the time of implantation. The 7- to 8-day embryoid body contains, in addition to endodermal and ectodermal derivatives, cells of the early mesoderm (Robertson, 1987). Developmental changes in the embryoid body morphology during differentiation can be seen clearly in the scanning electron microscope. Whereas ‘2 d’ embryoid bodies contained aggregates of nearly uncommitted ES cells, in the ‘5 d’ and even more in the ‘7 d’ embryoid bodies flat endodermal cells are developed on the surface of the embryoid body (Fig. 4AC). The ‘7 d’ embryoid bodies (treated with RA during different times) were plated and the formation of differentiated cell types was evaluated during further
485
cardiomyocyte differentiation (Fig. 4; Wobus et al., 1994b). ES cells treated during the first 2 days of embryoid body differentiation (‘0 to 2 d’) with high concentrations of RA resulted in a strong inhibition of cardiogenesis. The same concentrations of RA, when added between day 5 and 7 of embryoid body development (‘5 to 7 d’), however, induced no inhibition, but, in contrast, an induction of cardiomyocyte differentiation. ES cell-derived embryoid bodies cultivated in the presence of high RA concentrations between day 2 and 5 differentiated into skeletal myocytes instead into cardiomyocytes. This was demonstrated morphologically by immunofluorescence and RT-PCR analysis by time- and concentration-dependent inhibition of transcription of cardiac-specific myosin heavy chain (MHC) genes and the induction of transcription of skeletal musclespecific myogenin (Fig. 4). Furthermore, using the whole-ceil patch-clamp technique, we could demonstrate the functional activity of the induced myocytes by expression of skeletal muscle-specific CaZ+ channels and nicotinic cholinoceptor-operated channels. In summary, we may conclude that RA treatment during ectodermal (or neuroectodermal) differentiation (‘2 to 5 d’) resulted in a suppression of genes responsible for cardiomyocyte differentiation and in activation of genes involved in myogenesis. This stage-specific effect would imply that RA receptors (RARs) are present on embryoid bodies at these specific stages. In mice, the RAR subtypes a, /?and y were found to be specifically expressed in presomitic stage embryos in the lateral regions, during crest cell migration, and in the primitive streak region throughout the period of neurulation, respectively (Ruberte et al., 1991). Differentiating teratocarcinema cells showed significant alterations in the expression pattern of the RAR a, j and y subtypes especially during neuroectodermal differentiation: a strong up-regulation of RAR a and /I, and a rapid
down-regulation of RAR y mRNA was found suggesting a role for RAR a and fl expression during neuroectodermal differentiation (Jonk et al., 1992; Kruyt et al., 1991). As ES cells differentiating in embryoid bodies apparently reflect the specific time- and concentration-dependent effects of RA on embryonic development this ES cell-differentiation system provides a new model to study embryonic development under defined conditions in vitro and investigate the influence of exogenous factors during early embryogenesis for in vitro embryotoxicological studies.
Acknowlednemenrs-This
research was suDnorted by the
Deutsche sorsehungsgemeinschaft, the M&i&y of Science and Research of Sachsen-Anhalt. ZEBET. Robert-KochInstitute of Germany, Fonds der dhemischen Industrie and BAYER, Wuppertal, Gennany. We thank Dr Klaus Adler, IPK Gatersleben, for scanning electron microscopy of embryoid bodies and Dr Michael Wiles, Base1 Institute of Immunology, for submitting unpublished data.
486
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