Severe Inhibition of in Vitro Cardiomyogenesis in Mouse Embryonic Stem CellsEctopically Expressing EGAM1C Homeoprotein

Momoe IHA,1 Miki SOMA,1 Sho SATO,1 Yuki MORI,1 Saiko SUGAWARA,1 Kano KASUGA,2

Ikuo KOJIMA,2 Satoshi YAMADA,3 Shujiro SAKAKI,4 and Masayuki KOBAYASHI1;y

1Laboratory for Advanced Animal Cell Technology, Graduate School of Bioresource Sciences,Akita Prefectural University, Akita 010-0195, Japan2Laboratory for Biological Chemistry, Graduate School of Bioresource Sciences, Akita Prefectural University,Akita 010-0195, Japan3Tsukuba Corporate Research Laboratory, NOF Corporation, Tsukuba 300-2635, Japan4Applied Chemistry Department, Akita National College of Technology, Akita 011-8511, Japan

Received February 27, 2012; Accepted March 19, 2012; Online Publication, July 7, 2012

[doi:10.1271/bbb.120146]

Embryoid bodies were prepared from mouse embry-onic stem cells expressing exogenous EGAM1C toanalyze their ability to differentiate toward terminallydifferentiated cell types. The generation of cardiomyo-cytes was severely suppressed in Egam1c transfectantswithout upregulation of Nkx2-5, a crucial gene forcardiomyogenesis. These results indicate that EGAM1Cis capable of affecting terminal differentiation in mouseembryonic stem cells.

Key words: cardiomyogenesis; Crxos1 tv2; Egam1c;embryonic stem cells; Nkx2-5

Mouse embryonic stem (ES) cells are derived fromthe inner cell mass of blastocysts and are maintainedin vitro as a pluripotent self-renewing population in thepresence of leukemia inhibitory factor (LIF).1) WhenLIF is removed, cells undergo apparently spontaneousin vitro differentiation with commitment to a range ofembryological lineages.2) Hence, the ES cell-culturesystem is useful for the characterization of functionsof genes involved in pluripotency and differentiation.Furthermore, there is considerable interest in theprospect of exploring the ability to generate multiplecell types from ES cells to provide a renewable source ofmaterial for therapeutic cell transplantation in regener-ative medicine.

Recently, we identified Egam1c (GenBank accessionno. AB472694, also known as Crxos1 transcript variant2, NM 001145190) mRNA, which encodes the homeo-protein EGAM1C, in preimplantation mouse embryosand mouse ES cells.2,3) EGAM1C was capable ofaffecting hallmarks of ES cells.3) The exogenousexpression of EGAM1C stabilizes an undifferentiatedstate in the presence of LIF, while in the absence of LIF,it probably promotes differentiation toward cell typescomprising preimplantation embryos and embryos im-mediately after implantation, including epiblast, meso-derm, primitive endoderm, and trophectoderm. In vitrodifferentiation of ES cells is not restricted to theseprogenitor cells that arise in early embryogenesis, butalso gives rise to terminally differentiated cell types. The

effects of EGAM1C on the differentiation of mouse EScells prompted us to hypothesize that EGAM1C is alsocapable of affecting terminal differentiation. To test thispossibility, cardiomyocyte-differentiation from ES cellsby the generation of embryoid bodies (EBs) wasselected, because the procedure4,5) and the molecularmechanisms underlying cardiomyogenesis6) are wellestablished.Upon transfection of an Egam1c expression vector

driven by the CAG promoter,7) feeder-free mouse ESMG1.19 cells stably expressing EGAM1C at a levelsimilar to that in preimplantation embryos (clones C4and C28) were established as described previously,3) aswell as the isolation of control transfectants with anempty vector (clones E2 and E8). These transfectantswere routinely maintained on gelatin-coated plates inGlasgow Modified Eagle’s Medium (Sigma, St. Louis,MO) supplemented with 10% fetal calf serum (definedfor ES cells, Biological Industries, Haemek, Israel),puromycin (2 mg/mL), G418 (250 mg/mL), and re-combinant human LIF (made in-house, þLIF medium)at 37 �C in 5% CO2/air. Aliquots of the cell suspension(4,000 cells/200 mL) were transferred to a 96-well plate(U bottom, Lipidure-Coat Plate A-U96, NOF, Tsukuba,Japan) with puromycin (0.2 mg/mL) and G418 (25 mg/mL) in the absence of LIF (�LIF medium). The cellsuspension was cultured for 5d at 37 �C in 5% CO2/airto form EBs. Subsequently, the EBs were transferred toa 24-well plate coated with gelatin (four EBs/well forprotein or RNA analysis, and one EB/well for micro-scopic observation of its contraction) for attachmentcultures to promote cardiomyogenesis.5) The attachmentculture was continued for 7d. Microscopic observationwas performed daily to detect the generation ofcontracting cell populations derived from a single EBin each well. Extraction of total RNA and cDNAsynthesis were performed as reported previously.8)

Quantitative (q) RT-PCR reactions were performed induplicate using a QuantiTect SYBR Green PCR kit(Qiagen, Hilden, Germany) and a CFX96 Real-TimeDetection system (Bio-Rad, Hercules, CA) following themanufacturer’s protocols. Hydroxymethylbilane syn-

y To whom correspondence should be addressed. Fax: +81-18-872-1676; E-mail: makoba@akita-pu.ac.jp

Biosci. Biotechnol. Biochem., 76 (7), 1410–1412, 2012

Note

thase (Hmbs) was analyzed as a housekeeping gene. Theprimers for qRT-PCR were as follows: 50-GCTGACC-ACACAAGCAGAGAGGTT-30 and 50-TGGGATGGA-GAGTGGGCTGT-30 for ventricle-specific myosin lightchain-2 (Myl2), 50-TGGGCTGGCTGGAAAAGAAC-30

and 50-TCCCGGTGGAGAGCAGACAC-30 for cardiac�-myosin heavy chain (Mhc6), 50-CCGCCCCCACA-TTTTACC-30 and 50-ATCCGTCTCGGCTTTGTCC-30

for Nkx2-5, and 50-TCCCAGTGTCCAGCCATAAC-30

and 50-ATCTCCGCCCATCAGACC-30 for Mef2c. Theother primers were as in our previous study.3) The ratioof the number of cDNA copies for each gene to that forHmbs is indicated as expression levels. The resultsobtained by qRT-PCR analysis were subjected to one-way analysis of variance (ANOVA), followed by theTukey-Kramer multiple comparison test. Specific cellu-lar proteins were detected in two separate Western blottrials.3) The primary antibodies used were as follows;anti-EGAM1C9) (1:20,000), anti-NKX2-5 (1:10,000,sc-8697, Santa Cruz Biotechnology, Santa Cruz, CA),anti-MEF2C (1:10,000, sc-13266, Santa Cruz Biotech-nology), and anti-ACTB (�-ACTIN, 1:20,000, IMG-5142A, Imgenex, San Diego, CA).

As shown in Fig. 1A, ectopic expression of EGAM1Cwas detected in the EBs transfected with the Egam1c

expression vector; the expression level was evaluated as5- to 9-fold of the control EBs. In the EBs expressingEGAM1C, cell contraction was almost undetectable by7d of attachment culture, while it was observed in morethan 85% of the control EBs (Fig. 1B). Under theseconditions, the expression levels of mRNAs for Myl2and Myc6, typical cardiomyocyte markers, were almostentirely repressed in the EGAM1C-expressing EBs(Fig. 1C). These results indicate clearly that cardiomyo-genesis from ES cells was severely suppressed in theEgam1c transfectants.Next, the expression of Nkx2-510) and Mef2c,11) genes

encoding crucial transcription factors for cardiomyo-genesis, and their encoded proteins were analyzed. Inthe EBs expressing EGAM1C, the cellular content ofNKX2-5 decreased to 0.1- to 0.3-fold of the control(Fig. 2A), while that of MEF2C was maintained atalmost the original level. The expression level ofmRNAs for Nkx2-5 and Mef2c corresponded well withthat of their encoded proteins (Fig. 2B). During cardi-omyogenesis, transcription factors NKX2-5 and MEF2Cinteract physically and functionally and act as mutualcofactors. Hence they are considered to be critical forthe initiation and progression of cardiomyogenesis.6) Itis probable that the generation of cardiomyocytes fromES cells expressing EGAM1C was inhibited by impair-ment of the expression of Nkx2-5 by unknown mech-

A

B

C

Fig. 1. Effect of EGAM1C on Generation of Cardiomyocytes inMouse ES Cells.Embryoid bodies (EBs) were generated from ES cell-suspensions

(control transfectants, E2 and E8; Egam1c transfectants, C4 andC28) and transferred to attachment cultures to promote cardiomyo-genesis, as described in ‘‘Materials and Methods.’’ A, Cellularcontent of EGAM1C in control and Egam1c transfectants after thegeneration of EBs. EGAM1C (17 kDa) was detected by Westernblotting after 4d of attachment culture. ACTB (42 kDa) was aloading control. B, Time-dependent changes in rates of contractingEBs. Data represent the sum of two independent trials, because theresults obtained from the trials matched well. In the respectivetransfectants, 22 to 24 EBs in total were examined. C, Expressionlevels of the mRNAs for Myl2 and Mhc6 in EBs during attachmentculture. The expression levels of Myl2 and Mhc6 as cardiomyocytemarkers were quantified by qRT-PCR after 6d of attachment culture,and were normalized to that of Hmbs. Data are expressed as mean�SD (n ¼ 2). The mean value of the control transfectants was set as 1.�p < 0:05; ��p < 0:01.

A

B

C

Fig. 2. Expression Levels of Transcription Factors Involved inCardiomyogenesis in Mouse ES Cells.

The expression levels of the various mRNAs and their encodedproteins in EBs were determined after 4d of attachment culture. A,Cellular contents of NKX2-5 (39 kDa) and MEF2C (45 kDa) asdetermined by Western blotting. The data for ACTB are indicated inFig. 1A. B, Expression levels of mRNAs for Nkx2-5 and Mef2cquantified by qRT-PCR. Data are expressed as mean� SD (n ¼ 2).They were processed as described in the legend to Fig. 1C.��p < 0:01; NS, not significantly different. C, Expression levels ofmRNAs for Pou5f1 and Nanog quantified by qRT-PCR. Data areexpressed as mean� SD (n ¼ 2). They were processed as describedin the legend to Fig. 1C. The expression levels of these genes in anundifferentiated state sustained by LIF were almost the same in thesetransfectants. Hence the mean value during the undifferentiated statewas set as 1. �p < 0:05; ��p < 0:01.

EGAM1C and Inhibition of Cardiomyogenesis 1411

anisms. Many members of homeotic gene families act astranscriptional factors.12) Analysis of the relationshipbetween EGAM1C and the expression of Nkx2-5 andMef2c should indicate the role of EGAM1C as atranscription factor. Furthermore, we cannot rule outthe possibility that EGAM1C inhibits aberrant activationof Nkx2-5 in preimplantation mouse embryos. On theother hand, the expression levels of Pou5f1 (Oct4) andNanog, genes encoding essential transcription factors forthe maintenance of an undifferentiated state in EScells,12) in the EBs expressing EGAM1C were signifi-cantly below those in the control, and also substantiallylower than those in an undifferentiated state (Fig. 2C).Thus the Egam1c transfectants were confirmed to be in adifferentiated state. In our previous study,3) the expres-sion of specific marker genes Tpbpa for spongiotropho-blasts and Plat for the parietal endoderm was higher inEBs expressing EGAM1C. Thus EGAM1C probably ledto differentiation into extra-embryonic trophectodermand extra-embryonic endoderm lineages in EBs.

Taken together with our previous study,3) the ectopicexpression of EGAM1C is capable of affecting differ-entiation not only in lineages that arise early embryo-genesis, including epiblast, mesoderm, primitive endo-derm, and trophectoderm, but also in terminallydifferentiated cell types, such as cardiomyocytes, inthe ES cell-culture system.

Acknowledgments

We thank Dr. Hitoshi Niwa (RIKEN Center forDevelopmental Biology, Kobe, Japan) for providing

human LIF expressing vector. And we thank Dr. MaxGassmann (University of Zurich, Zurich, Switzerland)for providing pMGD20neo vector and MG1.19 cells.This study was supported in part by a research grantfrom the President of Akita Prefectural University toM.K.

References

1) Niwa H, Ogawa K, Shimosato D, and Adachi K, Nature, 460,

118–122 (2009).

2) Saito K, Abe H, Nakazawa M, Irokawa E, Watanabe M, Hosoi

Y, Soma M, Kasuga K, Kojima I, and Kobayashi M, Biol.

Reprod., 82, 687–697 (2010).

3) Iha M, Watanabe M, Kihara Y, Sugawara S, Saito K, Soma M,

Sato S, Mori Y, Kasuga K, Kojima I, Sasamura R, Murata J, and

Kobayashi M, Reproduction, 143, 477–489 (2012).

4) Desbaillets I, Ziegler U, Groscurth P, and Gassmann M, Exp.

Physiol., 85, 645–651 (2000).

5) Koike M, Kurosawa H, and Amano Y, Cytotechnology, 47, 3–

10 (2005).

6) Vincentz JW, Barnes RM, Firulli BA, Conway SJ, and Firulli

AB, Dev. Dyn., 237, 3809–3819 (2008).

7) Abe H, Nakazawa M, Kasuga K, Kojima I, and Kobayashi M,

Biotechnol. Biotech. Eq., 25, 2301–2304 (2011).

8) Soma M, Iha M, Sato S, Mori Y, Kasuga K, Kojima I, and

Kobayashi M, Biotechnol. Biotech. Eq., 25, 2679–2682 (2011).

9) Saito K, Ogawa A, Toyofuku K, Hosoi Y, Soma M, Iha M,

Kasuga K, Kojima I, and Kobayashi M, Reproduction, 141,

259–268 (2011).

10) Lints TJ, Parsons LM, Hartley L, Lyons I, and Harvey RP,

Development, 119, 419–431 (1993).

11) Edmondson DG, Lyons GE, Martin JF, and Olson EN,

Development, 120, 1251–1263 (1994).

12) Niwa H, Development, 134, 635–646 (2007).

1412 M. IHA et al.