Small Molecules that Induce Cardiomyogenesis in Embryonic Stem Cells

Article abstract of DOI:10.1021/ja038950i

A phenotypic cell-based screen of a large combinatorial chemical library led to the identification of a class of diaminopyrimidine compounds (cardiogenol A-D) which can selectively and efficiently induce mouse embryonic stem cells (ESCs) to differentiate into cardiomyocytes. ESC-derived cardiomyocytes were shown to express multiple cardiac muscle markers, including myosin heavy chain, GATA-4, MEF2, and Nkx2.5, and spontaneously form beating regions. Such small molecules will serve as useful chemical probes to study cardiac muscle differentiation and may ultimately facilitate the therapeutic application of ESCs for cardiac repair. Copyright

Full text of DOI:10.1021/ja038950i

Published on Web 01/22/2004
Small Molecules that Induce Cardiomyogenesis in Embryonic Stem Cells
Xu Wu,† Sheng Ding,† Qiang Ding,‡ Nathanael S. Gray,‡ and Peter G. Schultz*,†,‡
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, and Genomics Institute of the NoVartis
Research Foundation, 10675 John Jay Hopkins DriVe, San Diego, California 92121
Received October 8, 2003; E-mail: schultz@scripps.edu
Stem cells are multipotent cells with the ability to self-renew
and differentiate into specialized cells in response to appropriate
signals.1 Most tissues have endogenous stem/progenitor cells which,
upon injury to the organ, can proliferate and differentiate at the
damaged site. The adult heart is composed mainly of postmitotic
and terminally differentiated cells. Although a subpopulation of
myocardial cells with cardiac stem cell character was identified
recently, their limited availability hinders therapeutic applications.2
Stem cells derived from other tissues, such as bone marrow, have
been shown to be capable of repairing heart damage in animal
models,3 but inefficient differentiation and possible fusion with
Figure 1. A high throughput screen for cardiomyogenesis using an ANF-
somatic cells limit their use in cardiac repair.4 Pluripotent embryonic
promoter reporter assay.
stem (ES) cells represent a possible unlimited source of functional
cardiomyocytes. However, the in vitro differentiation of ES cells
into cardiomyocytes involves a poorly defined, inefficient, and
relatively nonselective process.5 Consequently, the development of
new approaches for the directed differentiation of ES cells into
cardiomyocytes will likely facilitate therapeutic application of ES
cells in heart disease, as well as provide important tools for probing
the molecular mechanism of cardiomyocyte differentiation and heart
development.
Recently, several small organic molecules identified in pheno-
typic cellular screens of combinatorial libraries of “privileged”
heterocycles6 were shown to be capable of inducing the selective
in vitro differentiation of both adult stem cells and embryonic stem
cells into osteoblasts and neurons.7,8 We therefore decided to screen
this same library for molecules that would selectively and efficiently
induce the differentiation of murine ESCs to cardiomyocytes. The
mouse embryonic carcinoma (EC) cell line P19 was initially chosen
for high throughput screening. P19 cells, like ESCs, are pluripotent
and can differentiate into cardiomyocytes after aggregation, forma-
tion of embryoid bodies (EBs), and treatment with 1% DMSO.9
Moreover, P19 cells are easy to culture, amenable to genetic
manipulation, and have a low frequency of spontaneous cardiac
differentiation.
Figure 2. Immunostaining of cardiac muscle markers in ESCs (A-E) and
P19CL6 cells (F) treated with 0.25 µM cardiogenol C: (A) and (F) myosin
heavy chain (green); (B) GATA-4 (red); (C) MEF2 (red); (D) Nkx2.5 (red);
and (E) myosin heavy chain (green) and MEF2 (red). Cell nuclei were
stained with DAPI (blue).
To develop an appropriate reporter assay, the promoter region
(∼700 kb) of the rat atrial natriuretic factor (ANF) gene10 was
cloned and inserted upstream of the luciferase gene in the PGL3-
BV reporter plasmid.11 ANF is a polypeptide hormone that is
synthesized primarily in cardiac myocytes and is a downstream
target of several cardiomyogenesis transcriptional factors; it is
considered a specific cardiomyocyte “marker” gene.12 A stable P19
clone harboring the reporter plasmid afforded a 5- to 7-fold increase
(Figure 1) in luciferase signal upon standard cardiomyogenesis
differentiation conditions for P19 cells (EB formation and treatment
with 1% DMSO). This cell line was used to screen a 100 000
compound heterocycle library in a monolayer format, and ap-
proximately 80 compounds were identified that up-regulated
luciferase activity >4-fold in the absence of EBs. Sarcomeric
myosin heavy chain (MHC) is one of the essential motor proteins
responsible for cardiac muscle contractibility5 and was used as a
secondary assay for differentiation. Thirty-five of the 80 compounds
also induced MHC expression in P19CL6 cells13,14 as determined
by immunostaining with anti-MHC antibody (MF20) (Figure 2F).
Among the latter compounds, four diaminopyrimidines, cardiogenol
A-D (Table 1), were the most potent in inducing MHC expression.
These compounds share significant structural similarities. All have
a 2-hydroxylamino substitution at the C-4 position, and all have
bulky, hydrophobic groups at the C-2 position, suggesting that they
function by a common mechanism.
To confirm that these compounds are general cardiomyogenesis
inducing agents, we analyzed their effects on undifferentiated R1
† The Scripps Research Institute.
‡ Genomics Institute of the Novartis Research Foundation.
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10.1021/ja038950i CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
of multiple cardiac muscle specific genes21,22 (e.g., myosin light
chain 2V, atrial natriuretic factor, eHAND, and homeodomain
transcription factor HOP); targeted interruption of Nkx2.5 gene is
embryonic lethal and results in arrest of cardiac development.23
Approximately 90% of cardiogenol C treated cells stain positive
for MEF2 and Nkx2.5, further confirming that ESCs are differenti-
ated into cardiac muscle by cardiogenol C.
Table 1. Chemical Structures and Biological Activities of
Cardiogenols
We are currently carrying out biochemical and genomics
experiments to identify the molecular targets of cardiogenol C.
These experiments may reveal novel molecular mechanisms related
to cardiomyogenesis, and ultimately facilitate the application of
ESCs to the repair of damaged myocardium in acute heart diseases.
Acknowledgment. This work was supported by the Novartis
Research Foundation and the Skaggs Institute for Chemical Biology
(P.G.S.). We thank Dr. Michael Schneider and Dr. Nakamura
Teruya for providing P19CL6 cells, and Ms. Mary Andahazy for
providing mouse ESCs (R1 cell line). This is manuscript number
16142-CH of the Scripps Research Institute.
Supporting Information Available: Detailed experimental pro-
cedures and compound characterization (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
mouse ESCs, which can be maintained in a pluripotent state with
the addition of leukemia inhibitory factor (LIF) in the culture
medium. R1 cells were plated in a monolayer in gelatin-coated 384-
well or 96-well plates in the absence of LIF and in the presence of
0.25 µM of compounds. After 7 days (3 days with compounds and
then 4 days without compounds), the presence of beating cardiac
muscle was visualized under microscope. In addition to the
expression of MHC (Figure 2A), the cardiac specific gene, GATA-
4, was detected by immunofluorescent staining using anti-GATA4
antibody (Figure 2B). GATA-4 is a transcription factor restricted
to developing heart, and its overexpression enhances cardiomyo-
genesis in P19 cells.15-17 Neither MHC nor GATA-4 is expressed
in undifferentiated R1 mouse ESCs. It was also observed that
compound treatment slowed cellular proliferation with no significant
cell death, indicating that this process is not simply a selection for
cardiac precursor cells with the death of cells in other lineages.
Cardiogenol C,18 which has a p-methoxy aniline substituent at
the pyrimidine C2 position, is the most potent compound with an
EC50 of 0.1 µM for inducing the differentiation of MHC positive
cardiomyocytes from ESCs. Cardiogenol C showed significant
cellular toxicity only at concentrations greater than 25 µM; after
treating R1 cells 0.25 µM compound for 3 days and further culturing
in medium without compound for 4 days, more than 50% of the
cells stained positive for MHC and more than 90% of the cells are
positive for GATA-4, consistent with the previous observation that
GATA-4 is expressed earlier than MHC.5 Moreover, there were
many beating areas in R1 cells treated with cardiogenol C,
suggesting that these MHC positive cells can form functional cardiac
muscle. These results indicate that the majority of the cell population
was induced by cardiogenol C to differentiate into cardiac lineage
(in the absence of aggregation and EB formation). This is in contrast
to the current standard method of inducing cardiomyogenesis of
ESCs by aggregation and formation of EBs, which results in only
5% of the cell population forming cardiomyocytes.5
References
(1) Spradling, A.; Drummond-Barbosa, D.; Kai, T. Nature 2001, 414, 98-
104.
(2) Beltrami, A. P.; Barlucchi, L.; Torella, D.; Baker, M.; Limana, F.;
Chimenti, S.; Kasahara, H.; Rota, M.; Musso, E.; Urbanek, K.; Leri, A.;
Kajstura, J.; Nadal-Ginard, B.; Anversa, P. Cell 2003, 114, 763-776.
(3) Ferrari, G.; Cusella-De Angelis, G.; Coletta, M.; Paolucci, E.; Stornaiuolo,
A.; Cossu, G.; Mavilio, F. Science 1998, 279, 1528-1530.
(4) Terada, N.; Hamazaki, T.; Oka, M.; Hoki, M.; Mastalerz, D. M.; Nakano,
Y.; Meyer, E. M.; Morel, L.; Petersen, B. E.; Scott, E. W. Nature 2002,
416, 542-545.
(5) Boheler, K.; Czyz, J.; Tweedie, D.; Yang, H.-T.; Anisimov, S. V.; Wobus,
A. M. Circ. Res. 2002, 91, 189-201.
(6) Ding, S.; Gray, N. S.; Wu, X.; Ding, Q.; Schultz, P. G. J. Am. Chem.
Soc. 2002, 124, 1594-1596.
(7) Wu, X.; Ding, S.; Ding, Q.; Gray, N. S.; Schultz, P. G. J. Am. Chem.
Soc. 2002, 124, 14520-14521.
(8) Ding, S.; Wu, T. Y. H.; Brinker, A.; Peters, E. C.; Hur, W.; Gray, N. S.;
Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7632-7637.
(9) Skerjanc, I. S. Trends CardioVasc. Med. 1999, 9, 139-143.
(10) Seidman, C. E.; Wong, D. W.; Jarcho, J. A.; Bloch, K. D.; Seidman, J.
G. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4104-4108.
(11) Technical Manual No. 033; Promega Corp.: Madison, WI, 2002.
(12) Boer, P. H. Exp. Cell Res. 1993, 207, 421-429.
(13) The P19CL6 cell line is a subclone of P19 EC cells with higher potential
for cardiomyogenesis.
(14) Habara-Ohkubo, A. Cell Struct. Funct. 1996, 21, 101-110.
(15) Grepin, C.; Nemer, G.; Nemer, M. DeVelopment 1997, 124, 2387-2395.
(16) Charron, F.; Nemer, M. Cell DeV. Biol. 1999, 10, 85-91.
(17) Garg, V.; Kathiriya, I. S.; Barnes, R.; Schluterman, M. K.; King, I. N.;
Butler, C. A.; Rothrock, C. R.; Eapen, R. S.; Hirayama-Yamada, K.; Joo,
K.; Matsuoka, R.; Cohen, J. C.; Srivastava, D. Nature 2003, 424, 443-
447.
(18) Cardiogenol C. 1H NMR (400 MHz, DMSO): δ (ppm) 3.42 (m, 2H),
3.55(m, 2H), 3.72 (s, 3H), 6.21 (d, 1H, J ) 7.2), 6.97 (d, 2H, J ) 8.9),
7.41 (d, 2H, J ) 8.4), 7.66 (d, 1H, J ) 7.1), 8.93 (s, 1H), 10.07 (s, 1H).
High-resolution MS (MALDI-FTMS): Calculated [MH+] (C13H17N4O2)
261.1346, found 261.1342. See Supporting Information for more details.
(19) Edmondson, D. G.; Lyons, G. E.; Martin, J. F.; Olson, E. N. DeVelopment
1994, 1251-1263.
(20) Lin, Q.; Schwarz, J.; Bucana, C.; Olson, E. N. Science 1997, 276, 1404-
1407.
(21) Small, E. M.; Krieg, P. A. DeV. Biol. 2003, 261, 116-131.
(22) Shin, C. H.; Liu, Z. P.; Passier, R.; Zhang, C. L.; Wang, D. Z.; Harris, T.
M.; Yamagishi, H.; Richardson, J. A.; Childs, G.; Olson, E. N.Cell 2002,
110, 725-735.
To further characterize the activity of cardiogenol C, we
examined the expression of the cardiac muscle cell specific
transcription factors MEF2 and Nkx2.5 (Figure 2C and D).
Members of the MEF2 family are essential for muscle develop-
ment.19,20 Nkx2.5 together with GATA-4 regulates the expression
(23) Lyons, I.; Parsons, L. M.; Hartley, L.; Li, R.; Andrews, J. E.; Robb, L.;
Harvey, R. P. Genes DeV. 1995, 9, 1654-1666.
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