Study on Cardiomyocyte Differentiation Induced by ...

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Study on Cardiomyocyte Differentiation Induced by ECP

2012 September

Guoliang JIN

Graduate School of Natural Science and Technology

(Doctor Course)

OKAYAMA UNIVERSITY

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CONTENTS

General Introductionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip4

Chapter 1 Effect of Eosinophil cationic protein (ECP) on

the cardiomyocyte differentiation in P19CL6 cells helliphelliphelliphelliphelliphelliphelliphellip21


activation of FGF receptor 1 signaling during

cardiomyocyte differentiationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37

Chapter 3 Effect of ECP on the expression of genes correlated

with cardiomyocyte differentiation in P19CL6 cells helliphelliphelliphelliphelliphellip63

Concluding Remarkshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip88

Acknowledgmentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip89

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Introduction

The human eosinophil cationic protein (ECP) also known as RNase 3 is an eosinophil

secretion protein that is involved in innate immunity and displays antipathogen and

proinflammatory activities ECP is released during degranulation of eosinophils In human

ECP encoded by the RNase3 gene belongs to the RNase A superfamily ECP is a

single-chain zinc-containing protein with a molecular weight ranging from 16 to 22 kDa

The heterogeneity of the molecule is partially due to differences in glycosylation of three

potential sites in its amino acid chain

1 Molecular characteristics of ECP

Eosinophil cationic protein (ECP) generally speaking is a heterogeneous molecule

originating from activated eosinophil granulocytes At present as shown in figure 1 103

kinds of ECP or ECP similar proteins have been sequenced And most of them are from

mammalian especially from hominid

Fig1 Taxonomic group of organisms that ECP been sequenced (From

httpwwwncbinlmnihgovprotein)

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Eosinophil cationic protein (ECP) that from human eosinophil granules was first isolated

and characterized by Gleich at al [1] They isolated ECP and another basic protein

eosinophil-derived neurotoxin (EDN) by gel filtration and ion exchange chromatography

on heparin-Sepharose NH2-terminal amino acid sequences of EDN and ECP from residue

1 through residue 55 showed a high similarity (67 homology) They also estimated that

the molecular mass of human ECP was 18-21 kD depending on the degree of glycosylation

Then Rosenberg et al did molecular cloning for ECP and reported the full-length cDNA

sequence The complete cDNA clone contains a Kozak-like translation initiation sequence

an open reading frame and a polyadenylation signal with a 15-base spacer preceding the

poly A tail The amino acid sequence encoded by the ECP cDNA confirms the identity of

residues identified by Gleich et al The mature ECP polypeptide (without leader sequence)

contains 133 amino acids which is one fewer than that found in EDN with a sequence

similarity between the two polypeptides of 66 The calculated molecular mass of mature

ECP is 15 6 kD and pI of ECP is 108 [2]

ECP and EDN sequences showed marked similarities to RNase from various species And

the amino acid sequence of both proteins showed a striking similarity with pancreatic-type

ribonucleases As shown in figure 2 they kept the specific residues of the active site (Gln11

His12 Lys41 and His119 RNase A numbering) and the eight Cys residues that form four

disulphide bridges Accordingly ECP and EDN were grouped in the human ribonuclease

family and have also been referred to as ribonuclease (RNase) 2 and 3 respectively [3 4]

In 2000 ECP has been cloned heterologously overexpressed purified and crystallized by

Mallorqui-Fernandez et al Its crystal structure has been determined and refined using data

up to 175 Aring resolutions The molecule displays the α+ β folding topology typical for

members of the ribonuclease A superfamily [5]

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Fig2 Alignment of the amino acid sequences of ECP and the bovine pancreatic RNase A

The α-helices and β-strand of ECP are displayed as labelled dark rods and light arrows

respectively The topologically equivalent secondary structure elements in the enzymes with

known three-dimensional structures are shaded α-helices with dark grey background and

β-strands with light grey The upper umbering corresponds to ECP and the lower one to

RNase A (From Goretti et al 2000)

Table 1 Crystalized recombinant ECP for July 2012

Complex

crystalized together

Resolution PDB-code Reference

no 200 Aring 1QMT Biox et al 1999

no 175 Aring 1DYT Mallorqui-Fernandez et al 2000

2rsquo 5rsquo-ADP 240 Aring 1H1H Mohan et al 2002

sulfate anions 170 Aring - Biox et al 2012

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Fig 3 Three-dimensional structure of human eosinophil cationic protein From Research

Collaboratory for Structural Bioinformatics (RCSB) protein data bank (PDB) accession

code 1DYT

Until now four crystalized ECP has been reported As shown in table 1 All the ECP were

recombinant type expressed in E coli BL21 (DE3) cell Crystals were grown by the

vapour diffusion hanging drop method

2 Functional characteristics and application of ECP

ECP as one of eosinophil secretion proteins involved in the host immune defense response

[6 7] has only been found in eosinophils granules and has not been detected in other

human tissues [8] Eosinophils were identified more than hundred years ago however their

roles in homeostasis and in disease still remain unclear The most prominent feature of the

eosinophils is their large secondary granules In the case of inflammation and asthma there

are increased levels of ECP in the body Therefore ECP has been developed as a biomarker

for eosinophilic disease and quantified in biological fluids including serum [9-12] saliva

[13 14] bronchoalveolar lavage [15] and nasal secretions [16] ECP as a biomarker was

studied firstly in Bronchial-Asthma by Bjornsson et al Then lots of researchers focus on it

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elevated ECP levels have been found in activated asthma [9-11 14 15 17-22] and other

respiratory tract diseases such as allergic rhinitis and bacterial sinusitis [16] In recent years

researchers also found that ECP could be biomarkers in other diseases such as inflammatory

bowel diseases [23] collagenous colitis [24] atopic keratoconjunctivitis [25 26] and

toxocara infection [27]

On the other hand ECP as ribonuclease has very weak ribonuclease activity Compared

with EDN ECP shows 100-fold less activity than EDN against a yeast RNA substrate [2

28] With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed

significant but low activity 70- to 200-fold less than that of bovine RNase A [29]

The low ribonuclease activity was related to its substrate subsites Boix et al investigated

ribonuclease activity of ECP through kinetic study Poly(C) poly(U) uridylyl(3rsquo5rsquo)

adenosine (UpA) uridylyl(3rsquo5rsquo) guanosine (UpG) cytidylyl(3rsquo5rsquo) adenosine (CpA)

uridine 2rsquo3rsquo-cyclic monophosphate (Ugtp) cytidine 2rsquo3rsquo-cyclic monophosphate (Cgtp) and

oligouridylic acids (Up)nUgtp n= 1ndash4 were used as substrates and the kinetic parameters

were determined In comparison with bovine pancreatic RNase A reduced catalytic

efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism

may be caused by the differences in the multisubsites structure[30] Mallorqui-Fernandez et

al got consistent conclusion they crystallized ECP and found that the catalytic active site

residues are conserved with respect to other ribonucleases of the superfamily but some

differences appear at substrate recognition subsites which may account in part for the low

catalytic activity [5] In 2002 Mohan et al reported crystal structure of ECP in complex

with 2rsquo 5rsquo-ADP at 20 Aring resolution The study is the first detailed structural analysis of the

nucleotide recognition site in ECP Residues Gln-14 His-15 and Lys-38 make hydrogen

bond interactions with the phosphate at the P1 site while His-128 interacts with the purine

ring at the B2 site A new phosphate binding site P-1 has been identified which involves

Arg-34 [31]

Moreover ECP shows a wide range of biological activities in addition to its weak

ribonuclease activity ECPs biological activities related with eosinophils in most cases are

not associated with ribonuclease activity It has been shown to possess cytotoxic activity

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neurotoxic activity and antimicrobial activity against bacteria pathogens and fungi

antiviral function and fibrosis promoting functions [6 32-35] Young et al reported that

purified human ECP could damage schistosomula of Schistosoma mansoni at low

concentrations Membrane damage was mediated by ECP through forming transmembrane

pores [33] Sorrentino et al treated EDN liver RNase and ECP with iodoacetic acid at

pH 55 which resulted in inactivation of their RNase activity and also destroyed their

neurotoxicity So they concluded that RNase activity is necessary but not sufficient to

induce neurotoxic action[36] However Rosenberg got the different conclusion Mutant

recombinant ECP that have no ribonuclease activity still shows antibacterial activity He

believed that ribonuclease activity and cytotoxicity are in that case independent functions

of ECP[37]About antiviral activity Domachowske et al prepared recombinant human ECP

(rhECP) prepared in baculovirus The rhECP was N-glycosylated and had similar to

100-fold more ribonuclease activity than nonglycosylated rhECP prepared in bacteria The

enzymatic activity of rhECP was sensitive to inhibition by placental ribonuclease inhibitor

(RI) And they found that rhECP promotes ribonuclease-dependent toxicity toward

extracellular virions of the single-stranded RNA virus respiratory syncytial virus group B

(RSV-B) [34]

On the basis of structure analysis ECP variants modified at basic and hydrophobic residues

have been constructed The bactericidal activity of both native ECP and point-mutated

variants were tested against Escherichia coli and Staphylococcus aureus Changes in the

leakage of liposome vesicles suggests that basic amino acids play in addition to the effect

on the disruption of the cellular membrane other roles such as specific binding on the

surface of the bacteria cell[38] Navarro et al analyzed ECP cytotoxic activity on

eukaryotic cell lines They found that ECP effects begin with its binding and aggregation to

the cell surface and then induce cell-specific morphological and biochemical changes such

as chromatin condensation reversion of membrane asymmetry reactive oxygen species

production and activation of caspase-3-like activity and eventually cell death They also

reported that the ribonuclease activity component of ECP is not involved in cytotoxic

activity as no RNA degradation is observed [39] Sanchez et al applied enzymatic and

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chemical limited cleavage to search for active sequence determinants for antimicrobial

activity and confirm the main role of the protein N-terminal The results corroborated that

the 11-35 internal sequence and in particular the 28-35 stretch is essential The identified

region is involved in both ECP aggregation and bacteria envelope binding properties[40]

Torrent et al studied ECP aggregation they demonstrated that ECP is able to form amyloid

aggregates The amyloid protofibrils formed by ECP bind amyloid-diagnostic dyes as Th-T

and Congo Red present a fibrilar structure under electron microscopy They identified an

N-terminus hydrophobic patch (residues 8-16) that is required for the amyloid aggregation

process [41] Pulido et al found ECP has a remarkable affinity for lipopolysaccharide (LPS)

and a distinctive agglutinating activity By using a battery of LPS-truncated E coli mutant

strains they demonstrated that the polysaccharide moiety of LPS is essential for

ECP-mediated bacterial agglutination thereby modulating its antimicrobial action The

mechanism of action of ECP at the bacterial surface is drastically affected by the LPS

structure and in particular by its polysaccharide moiety They also analyzed an N-terminal

fragment that retains the whole protein activity and displays similar cell agglutination

behavior A fragment with further minimization of the antimicrobial domain though

retaining the antimicrobial capacity significantly loses its agglutinating activity exhibiting

a different mechanism of action which is not dependent on the LPS composition [42]

In 2012 Boix et al crystallized ECP in complex with sulfate anions in a new momoclinic

crystal form The study provides direct evidence of the main protein sulfate binding sites

Three main sites (S1-S3) are located in the protein active site involved in sulfated

heterosaccharide binding S1 and S2 overlap with the phosphate binding sites involved in

RNase nucleotide recognition A new site (S3) is one of the key anchoring points for

sulfated ligands In particular site S3 is unique to ECP in the RNase A superfamily This

may explaine ECP reduced ribonuclease activity and high affinity for glycosaminoglycans

[43]

Some researchers studied the effect of ECP on mammalian tumor cells Maeda et al

evaluated the effect of ECP on 13 mammalian cell lines They found that ECP inhibited the

growth of several cell lines including those derived from carcinoma and leukemia in a

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dose-dependent manner ECP significantly suppressed the size of colonies of A431 cells

and decreased K562 cells in G(1)G(0) phase However there was little evidence that ECP

killed cells in either cell line Those results suggest that growth inhibition by ECP is

dependent on cell type and is cytostatic [44] Similar studies were also done on gastric

cancer cells[45] oral malignant tumor cells[46] and Hodgkin lymphoma cell[47]

Very recently we have demonstrated that eosinophil cationic protein (ECP) affected the

development of cytoskeleton in normal fibroblast cells and the differentiation of rat

neonatal cardiomyocytes[48] This finding implies that ECP might be useful as a

cardiomyocyte differentiation factor in the repair of cardiac tissue by stimulating the

differentiation of cardiomyocyte progenitor cells[48]

3 Cardiomyocyte differentiation

Heart failure is currently the most common cardiac disease largely due to the increasing

average age of the population [49 50] Heart failure is a progressive disorder initiated by an

acute or gradual loss of functional cardiomyocytes resulting in diminished cardiac output

and cardiac performance for instance after myocardial infarction or in patients suffering

from hypertension Current therapies include lifestyle modification drug treatments

surgery and ultimately heart transplantation

Cardiac transplantation remains the only therapeutic option for end-stage heart failure but

the low number of organ donors and the side effects of immunosuppressive drugs limit the

access to a transplantation program to a few thousand patients a year [51 52] In this

context alternative approaches may include restoring heart function via induction of

endogenous regenerative processes and transferring progenitor cells to produce new

myocardium Cardiomyocytes are currently thought to be postmitotic cells that withdraw

from the cell cycle soon after birth Nevertheless mitoses in the myocyte population have

been detected in the human heart after myocardial infarction [53] Recent studies suggest

that cardiomyocytes may be continually replaced in the heart through processes involving

differentiation maturation senescence and death [54] Cardiomyocyte turnover in the

mammalian heart has been demonstrated experimentally and shown to be triggered by

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cardiac injury [55] Regenerative stem cell therapy is a relatively new frontier in the battle

against cardiovascular disease that has sparked intense research and criticism In theory

embryonic stem (ES) cells could be produced in a large numbers differentiated to

cardiomyocytes and then used as a renewable source for cardiac cell therapy [56] One of

the major obstacles hindering the clinical use of ES cells derived cardiomyocytes for

cardiac regenerative therapy is inefficient cardiac differentiation leading to insufficient

amount of cardiomyocytes [57] and the potential development of teratomas The most

common approach to solve these problems are to apply knowledge of developmental

biology in employing cardiogenesis-related factors for inducing stem cells cardiomyocyte

differentiation [58-60]

According to previous study ECP enhanced neonatal rat cardiomyocyte differentiation

However the role of ECP on the cardiomyocyte differentiation and detail mechanism were

still unclear These unclear problems provided me with motivation to initiate this study

This thesis consists of 3 chapters in addition to Introduction and Conclusions

In chapter 1 the functional role of ECP in the cardiogenesis was investigated by mouse

P19CL6 embryonic carcinoma cells ECP was confirmed to accelerate the cardiomyocyte

differentiation of P19CL6 cells by the rate and area size of beating and the expression of

cardiomyocyte specific genes

In chapter 2 I detected the molecular mechanism of ECP on the cardiomyocyte

differentiation of P19CL6 cells focus on FGFR signaling I concluded that ECP induced

mesoderm differentiation by stimulating FGF signaling pathway and then enhanced

subsequent cardiomyocyte differentiation in concert with DMSO in P19CL6 cells

In chapter 3 I checked effects of ECP on the other signaling pathways correlated with

cardiomyocyte differentiation such as Wntβ-catenin PI3KAkt EphA1 ET-1 and so on

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4 References

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Eosinophil-Derived Neurotoxin and Eosinophil Cationic Protein - Homology with

Ribonuclease Proceedings of the National Academy of Sciences of the United

States of America 1986 83(10) p 3146-3150

2 Rosenberg HF SJ Ackerman and DG Tenen Human Eosinophil Cationic

Protein - Molecular-Cloning of a Cyto-Toxin and Helminthotoxin with

Ribonuclease-Activity Journal of Experimental Medicine 1989 170(1) p

163-176

3 Beintema JJ The ribonuclease A superfamily - Introduction Cellular and

Molecular Life Sciences 1998 54(8) p 763-765

4 Beintema JJ and RG Kleineidam The ribonuclease A superfamily general

discussion Cellular and Molecular Life Sciences 1998 54(8) p 825-832

5 Mallorqui-Fernandez G et al Three-dimensional crystal structure of human

eosinophil cationic protein (RNase 3) at 175 angstrom resolution Journal of

Molecular Biology 2000 300(5) p 1297-1307

6 Boix E et al The antipathogen activities of eosinophil cationic protein Current

Pharmaceutical Biotechnology 2008 9(3) p 141-152

7 Venge P et al Eosinophil cationic protein (ECP) molecular and biological

properties and the use of ECP as a marker of eosinophil activation in disease

Clinical and Experimental Allergy 1999 29(9) p 1172-1186

8 Futami J et al Tissue-specific expression of pancreatic-type RNases and RNase

inhibitor in humans DNA and Cell Biology 1997 16(4) p 413-419

9 Sorva R et al Eosinophil cationic protein in induced sputum as a marker of

inflammation in asthmatic children Pediatric Allergy and Immunology 1997 8(1)

p 45-50

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10 Niimi A et al Serum eosinophil cationic protein as a marker of eosinophilic

inflammation in asthma Clinical and Experimental Allergy 1998 28(2) p

233-240

11 Kang H et al Serum Eosinophil Cationic Protein a Useful Follow-up Marker in

Bakers Asthma Journal of Allergy and Clinical Immunology 2009 123(2) p

S235-S235

12 Guilpain P et al Serum eosinophil cationic protein - A marker of disease activity

in Churg-Strauss syndrome Autoimmunity Pt C 2007 1107 p 392-399

13 Kurklu E and K Guven Eosinophil cationic protein in saliva a marker for disease

activity in oral lesions Oral Diseases 2010 16(6) p 540-540

14 Schmekel B et al Eosinophil cationic protein (ECP) in saliva a new marker of

disease activity in bronchial asthma Respiratory Medicine 2001 95(8) p 670-675

15 Robinson DS et al Eosinophil Cationic Protein (Ecp) and Eosinophil-Derived

Neurotoxin (Edn) Levels Are Elevated in Blood and Bronchoalveolar Lavage from

Asthmatics Journal of Allergy and Clinical Immunology 1994 93(1) p 228-228

16 Rasp G et al IL-5 IgE ECP and ICAM-1 in nasal secretions of patients suffering

from chronic non-allergic sinusitis allergic rhinitis and non-allergic nasal

polyposis Journal of Allergy and Clinical Immunology 1999 103(1) p

S247-S247

17 Terada A et al Eosinophil Cationic Protein (Ecp) Is a Useful Marker for

Differential-Diagnosis of Wheezing in Children Journal of Allergy and Clinical

Immunology 1995 95(1) p 277-277

18 McGill KA et al Use of eosinophil cationic protein as a predictive marker for

exacerbations of asthma Journal of Allergy and Clinical Immunology 1996 97(1)

p 511-511

19 Vatrella A et al Serum eosinophil cationic protein (ECP) as a marker of disease

activity and treatment efficacy in seasonal asthma Allergy 1996 51(8) p 547-555

20 Terada A et al Serum eosinophil cationic protein as a guiding marker for

initiation of inhaled corticosteroid in children with asthma Journal of Allergy and

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Clinical Immunology 1997 99(1) p 1636-1636

21 Iikura Y et al Evaluation of serum eosinophil cationic protein (ECP) monitoring

childhood asthma ECP as a marker for indication and discontinuation of inhaled

steroid therapy Journal of Allergy and Clinical Immunology 1998 101(1) p

S8-S8

22 Bystrom J et al Dissecting the role of eosinophil cationic protein in upper airway

disease Current Opinion in Allergy and Clinical Immunology 2012 12(1) p

18-23

23 Dainese R et al Role of serological markers of activated eosinophils in

inflammatory bowel diseases European Journal of Gastroenterology amp Hepatology

2012 24(4) p 393-397

24 Wagner M et al Fecal eosinophil cationic protein as a marker of active disease

and treatment outcome in collagenous colitis A pilot study Scandinavian Journal of

Gastroenterology 2011 46(7-8) p 849-854

25 Wakamatsu TH et al IgE and eosinophil cationic protein (ECP) as markers of

severity in the diagnosis of atopic keratoconjunctivitis British Journal of

Ophthalmology 2012 96(4) p 581-586

26 Wakamatsu TH et al Eosinophil cationic protein as a marker for assessing the

efficacy of tacrolimus ophthalmic solution in the treatment of atopic

keratoconjunctivitis Molecular Vision 2011 17(103) p 932-938

27 Magnaval JF et al Eosinophil cationic protein as a possible marker of active

human Toxocara infection Allergy 2001 56(11) p 1096-1099

28 Gullberg U et al The Cytotoxic Eosinophil Cationic Protein (Ecp) Has

Ribonuclease-Activity Biochemical and Biophysical Research Communications

1986 139(3) p 1239-1242

29 Sorrentino S and DG Glitz Ribonuclease-Activity and Substrate Preference of

Human Eosinophil Cationic Protein (Ecp) Febs Letters 1991 288(1-2) p 23-26

30 Boix E et al Kinetic and product distribution analysis of human eosinophil

cationic protein indicates a subsite arrangement that favors exonuclease-type

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activity Journal of Biological Chemistry 1999 274(22) p 15605-15614

31 Mohan CG et al The crystal structure of eosinophil cationic protein in complex

with 25-ADP at 20 A resolution reveals the details of the ribonucleolytic active

site Biochemistry 2002 41(40) p 12100-6

32 Meyer JS and JM Foley The Encephalopathy Produced by Extracts of

Eosinophils and Bone Marrow Journal of Neuropathology and Experimental

Neurology 1953 12(4) p 349-362

33 Young JD et al Mechanism of Membrane Damage Mediated by Human

Eosinophil Cationic Protein Nature 1986 321(6070) p 613-616

34 Domachowske JB et al Eosinophil cationic protein RNase 3 is another

RNaseA-family ribonuclease with direct antiviral activity Nucleic Acids Research

1998 26(14) p 3358-3363

35 Torrent M et al Topography studies on the membrane interaction mechanism of

the eosinophil cationic protein Biochemistry 2007 46(3) p 720-733

36 Sorrentino S et al Eosinophil-Derived Neurotoxin and Human Liver

Ribonuclease - Identity of Structure and Linkage of Neurotoxicity to Nuclease

Activity Journal of Biological Chemistry 1992 267(21) p 14859-14865

37 Rosenberg HF Recombinant Human Eosinophil Cationic Protein -

Ribonuclease-Activity Is Not Essential for Cytotoxicity Journal of Biological

Chemistry 1995 270(14) p 7876-7881

38 Carreras E et al Both aromatic and cationic residues contribute to the

membrane-lytic and bactericidal activity of eosinophil cationic protein

Biochemistry 2003 42(22) p 6636-6644

39 Navarro S et al The cytotoxicity of eosinophil cationic proteinribonuclease 3 on

eukaryotic cell lines takes place through its aggregation on the cell membrane

Cellular and Molecular Life Sciences 2008 65(2) p 324-337

40 Sanchez D et al Mapping the eosinophil cationic protein antimicrobial activity by

chemical and enzymatic cleavage Biochimie 2011 93(2) p 331-338

41 Torrent M et al Eosinophil Cationic Protein Aggregation Identification of an

- 17 -

N-Terminus Amyloid Prone Region Biomacromolecules 2010 11(8) p

1983-1990

42 Pulido D et al Antimicrobial Action and Cell Agglutination by the Eosinophil

Cationic Protein Are Modulated by the Cell Wall Lipopolysaccharide Structure

Antimicrobial Agents and Chemotherapy 2012 56(5) p 2378-2385

43 Boix E et al The sulfate-binding site structure of the human eosinophil cationic

protein as revealed by a new crystal form Journal of Structural Biology 2012

179(1) p 1-9

44 Maeda T et al Growth inhibition of mammalian cells by eosinophil cationic

protein European Journal of Biochemistry 2002 269(1) p 307-316

45 Koo JW et al Association of of RNase3 Polymorphisms with the Susceptibility

of Gastric Cancer Journal of the Korean Surgical Society 2010 78(5) p 283-289

46 Pereira MC DT Oliveira and LP Kowalski The role of eosinophils and

eosinophil cationic protein in oral cancer A review Archives of Oral Biology 2011

56(4) p 353-358

47 Glimelius I et al Effect of eosinophil cationic protein (ECP) on Hodgkin

lymphoma cell lines Experimental Hematology 2011 39(8) p 850-858

48 Fukuda T et al Human eosinophil cationic protein enhances stress fiber formation

in Balbc 3T3 fibroblasts and differentiation of rat neonatal cardiomyocytes Growth

Factors 2009 27(4) p 228-236

49 P Hodges Heart failure epidemiologic update Crit Care Nurs Q 32 (2009)

24ndash32

50 Y Sun Myocardial repairremodelling following infarction roles of local factors

Cardiovasc Res 81 (2009) 482ndash490

51 DO Taylor LB Edwards P Aurora JD Christie F Dobbels R Kirk AO

Rahmel AY Kucheryavaya MI Hertz Registry of the International Society for

Heart and Lung Transplantation twenty-fifth official adult heart transplant

reportmdash2008 J Heart Lung Transplant 27 (2008) 943ndash956

52 JG Augoustides H Riha Recent progress in heart failure treatment and heart

- 18 -

transplantation J Cardiothorac Vasc Anesth (2009)

53 AP Beltrami K Urbanek J Kajstura SM Yan N Finato R Bussani B

Nadal-Ginard F Silvestri A Leri CA Beltrami P Anversa Evidence that

human cardiac myocytes divide after myocardial infarction N Engl J Med 344

(2001) 1750ndash1757

54 P Anversa B Nadal-Ginard Myocyte renewal and ventricular remodelling Nature

415 (2002) 240ndash243

55 PC Hsieh VF Segers ME Davis C MacGillivray J Gannon JD Molkentin J

Robbins RT Lee Evidence from a genetic fate-mapping study that stem cells

refresh adult mammalian cardiomyocytes after injury Nat Med 13 (2007)

970ndash974

56 Habara-Ohkubo A 1996 Differentiation of beating cardiac muscle cells from a

derivative of P19 embryonal carcinoma cells Cell Struct Funct 21101-110

57 Segers VF Lee RT 2008 Stem-cell therapy for cardiac disease Nature 451937-942

58 Laflamme MA Chen KY Naumova AV Muskheli V Fugate JA Dupras SK

Reinecke H Xu C Hassanipour M Police S OSullivan C Collins L Chen Y

Minami E Gill EA Ueno S Yuan C Gold J Murry CE 2007 Cardiomyocytes

derived from human embryonic stem cells in pro-survival factors enhance function

of infarcted rat hearts Nat Biotechnol 251015-1024

59 Singh AM Li FQ Hamazaki T Kasahara H Takemaru KI Terada N 2007 Chibby

an antagonist of the Wntbeta-catenin pathway facilitates cardiomyocyte

differentiation of murine embryonic stem cells Circulation 115617-626

60 Yuasa S Itabashi Y Koshimizu U Tanaka T Sugimura K Kinoshita M Hattori F

Fukami S Shimazaki T Okano H Ogawa S Fukuda K 2005 Transient inhibition

of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse

embryonic stem cells Nature Biotechnology 23897-897

- 19 -

Chapter 1

Effect of Eosinophil cationic protein (ECP) on the

cardiomyocyte differentiation in P19CL6 (mouse

teratocarcinoma) cells

- 20 -

Abstract

We previously demonstrated that eosinophil cationic protein (ECP) promoted the

differentiation of the rat primary cardiac cells Here we investigated the functional role of

ECP in the cardiomyogenesis using mouse P19CL6 embryonic carcinoma cells which are

well known as a model of cardiomyocyte differentiation in the presence of DMSO ECP

was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells by the rate

and area size of beating and the expression of cardiomyocyte specific genes such as

GATA-4 and α-MHC P19CL6 cells at 12 days of treatment were immunologically stained

with antibodies against actinin actin ANF and cardiac troponin I The wider size of

immunoreactive area was observed in the presence of ECP than that without ECP This is

the first to report the function of ECP on cardiomyocyte differentiation from stem cells in

vitro We are now trying to develop ECP as cardiomyocyte differentiation factor which

could be devoted in the development of novel therapies for cardiac regeneration

- 21 -

Introduction

Unlike other organs such as the skin and liver the heart is not able to regenerate sufficient

cardiomyocytes to undergo extensive repair Shortly after birth most cardiomyocytes stop

dividing and are terminally differentiated Ischemic heart disease and congestive heart

failure are major reasons of morbidity and mortality in the world and place a substantial

economic burden on most health systems [1] Heart failure occurs as the end result of

pathological remodeling of the myocardium in response to either ischemic or nonischemic

injury and a core component of this process is cardiomyocyte death and loss of myocardial

cell mass [2] Cardiac transplantation is a major treatment of choice for end-stage heart

failure at present However the number of available donor organs limits this treatment

option to a minority of patients [3] The development of new therapeutic paradigms for

heart failure and alternative therapies such as cardiac cell replacement has therefore

become imperative Stem cell therapy is a relatively new frontier in the battle against

cardiovascular disease that has sparked intense research and criticism In theory ES cells

could be produced in a large numbers differentiated into cardiomyocytes which could be

used as a renewable source for cardiac cell therapy [23] Over the past few years several

promising results have been reported but many hurdles remain before stem cells can

actually be used to treat patients with a damaged heartsuch as low efficiency of

differentiation find a inducer which can enhance cardiomyocyte differentiation is hot point

on this research

P19 embryonal carcinoma cells

P19 mouse embryonal carcinoma cell line has been reported to differentiate into an

embryonic cardiac muscle phenotype in vitro [23 24] upon the addition of dimethyl

sulfoxide (DMSO) Differentiated P19 cells have been reported to retain the ability to

spontaneously contract and shown to express transcripts in a temporal manner during

culture suggestive of a cardiac muscle phenotype [28-30] and as such these cells have

therefore been extensively used to study cardiac cell physiology [2425282931] although

- 22 -

with the caveat that these cells are embryonic instead However in addition to these cardiac

muscle specific properties P19 cells also display pluripotent properties and can be

differentiated into cells displaying either a skeletal muscle or neural phenotype [24 26-28]

There has thus been some concern about the homogeneity of DMSO differentiated P19

cultures with a heterogeneous cell population following differentiation significantly

reducing the utility of these cells as a cardiac-muscle-specific model There has thus been

much interest in identifying subclones of P19 cells that more robustly differentiate into

cardiomyocytes The P19CL6 cells line a sub-clone of P19 embryonal cells has been

reported to efficiently differentiate into beating cardiomyocytes upon exposure to DMSO

under adherent culture conditions [32] and has been widely used as an in vitro model of

cardiovascular cells [242533-39] It is clear that the P19CL6 cell line regards as a model

system for the study of cardiomyocyte development and differentiation In this chapter we

used this cell line to examine the function of ECP on the cardiomyocyte differentiation

Materials and Methods

Cell culture

P19CL6 cells which were obtained from RIKEN Bioresource Center Cell Bank Japan

were cultured essentially as described previously[13 14] Briefly the cells were grown in

100-mm tissue culture dishes under adherent conditions with α-minimum essential medium

(α-MEM) (Invitrogen Tokyo Japan) containing 10 fetal bovine serum (FBS)

(Invitrogen) as growth medium in a 5 CO2 atmosphere at 37degC To induce

cardiomyocyte differentiation under adherent conditions P19CL6 cells were plated at a

density of 5times105 in 60-mm tissue culture dishes in growth medium Twenty four hours later

the medium was replaced with growth medium containing 1 DMSO (Nacalai tesque

Kyoto Japan) as differentiation medium The medium was changed to every 2 days and the

cells were maintained under fresh conditions until they started beating

Reagents and antibodies

- 23 -

Recombinant human ECP was expressed in bacteria and prepared as described previously

[15 16] SU5402 which is an FGF receptor (FGFR) inhibitor was purchased from

Calbiochem (San Diego CA) Anti-actin rabbit polyclonal antibody anti-actinin mouse

monoclonal antibody and anti-cardiac troponin I mouse monoclonal antibody were from

Sigma-Aldrich

Reverse transcription polymerase chain reaction (RT-PCR) and real time qPCR

P19CL6 cells cultured under various conditions were harvested and total RNA was

extracted with RNeasy Mini Kit (QIAGEN MD) In order to exclude the contamination of

genomic DNA the extracted RNA was treated with RNase-Free DNase1 (Takara Japan)

Five micrograms of total RNA was then used to synthesize first-strand cDNA with

oligo-dT18 and 5 unitsμL of SuperScript Ⅱ (Invitrogen CA) in a reaction volume of 20 μL

following the manufacturerrsquos instructions For semiquantitative analysis reverse

transcribed products were pooled and fourfold serial dilutions were used for RT-qPCR

(Lyte-Cycler 480 Roche Diagnostics Germany) PCR was performed in a reaction volume

of 20 μL with 200 nM deoxynucleoside triphosphates 500 nM each of sense and antisense

primers and 5 units100 μL Taq polymerase (Takara Japan) The amplification reaction

was carried out in an authorized thermal cycler (Eppendorf Germany) The sequences of

primers used for the RT-PCR are following (forward and reverse) GATA4

5-ACTCTGGAGGCGAGATGGG-3 and 5-CTCGGCATTACGACGCCACAG-3

α-MHC 5-GGAAGAGTGAGCGGCGCATCAAGGA-3 and

5-TCTGCTGGAGAGGTTATTCCTCG-3 β-MHC

5-CGGAGGAGCAGGCCAACACCAACT-3 and

5-GCAAAGGCTCCAGGTCTGAGGGCTT-3 Nkx25

5-TGGCAGAGCTGCGCGCGGAGATG-3 and

5-CGTGGCTTCCGTCGCCGCCGTGC-3 glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) 5-CCCTTCATTGACCTCAACTAC-3 and

5-CCACCTTCTTGATGTCATCAT-3 PCR was performed for 1 cycle at 94degC for 5 min

followed by 25ndash35 cycles of denaturation at 94degC for 30 sec annealing at 56ndash62degC

- 24 -

depending on the melting temperature of each pair of primers for 30 sec followed by

extension at 72degC for 1 min

For quantitative analysis of gene expression levels real time qPCR were performed and the

data were normalized to GAPDH Primers used for the real time qPCR were as following

(forward and reverse) 5-AGCTCTCCAACCTATGCGGACAAT-3 and

5-TATCATGGGACTGCAGCATGGACA-3

GATA4 5-CAGCCCAGTCCTGCACAGCC-3 and

5-GGGCCGGTTGATGCCGTTCA-3 α-MHC

5-GCCATCACAGATGCCGCCATGA-3 and 5-TGCGCTTTTGCTCAGCCTCCA-3

Every PCR condition was confirmed to be within the linear range and within the

semiquantitative range for these specific genes and primer pairs To confirm that the

obtained bands were not derived from contaminated genomic DNA a negative assay was

performed on each sample without reverse transcriptase before PCR Amplified samples

were electrophoresed on 2 agarose gels and stained with ethidium bromide GAPDH

mRNA levels were need as an internal control

Immunostaining of P19CL6 cells

P19CL6 cells were seeded into 12-well tissue culture plate (TPP Switzerland) with a

sterilized cover slip at 105 cells per well in growth medium Twenty-four hours later the

medium was changed to differentiation medium with or without 1microgmL (66 nM) of ECP

On the 12th day the cells were washed with PBS twice fixed with 4 (wv)

paraformaldehyde at room temperature for 30 min permeabilized in phosphate-buffered

saline buffer containing 005 Tween-20 (PBST) and then blocked with PBST containing

5 BSA (blocking buffer) for one hour The cells were incubated with anti-actin and

anti-actinin antibodies diluted in blocking buffer at a dilution of 1200 at 4 for 16 h The

cells were washed twice with PBST and then incubated with both Alexa 488-conjugated

anti-mouse IgG antibody and Alexa Fluorreg 555-conjugated anti-rabbit IgG secondary

antibody (Amersham-Pharmacia) in blocking buffer at 25 ˚C for 1 h For cardiac troponin I

staining mouse anti-troponin I antibody was used at a dilution of 1200 Fluorescent

- 25 -

images of immunostaining were observed under a confocal laser microscope LSM510-V30

(Carl Zeiss UK) at 488 nm and 543 nm

Statistical analysis

All experiments were replicated at least three times Statistical data analysis was performed

using the Studentrsquos t-test by Excel software Values are reported as means plusmn SD The data

were considered statistically significant when p lt 005

Results

Enhanced cardiomyocyte differentiation of P19CL6 cells after ECP treatment

P19CL6 cells differentiate into cardiomyocyte in the presence of 1 DMSO

DMSO-treated P19CL6 cells are considered as a model of cardiomyocyte differentiation of

which the molecular mechanism has been extensively analyzed [17-19] Since we found

that ECP promoted the differentiation of cardiomyocytes in the rat neonate we tried to

assess the activity of ECP using P19CL6 cells First P19CL6 cells were cultured in the

presence or absence of ECP and DMSO The medium was changed every two days and

then morphology of cells was observed under microscope On the 8th day the cells

spontaneously started beating in the presence of both ECP and DMSO while it took 12 days

for the cells to start beating with only DMSO (Fig 1) ECP was found to accelerate

cardiomyocyte differentiation by almost 4 days On day 12 the beating rate of the cells was

50plusmn8 timesmin in the presence of ECP and 30plusmn10 timesmin without ECP (Fig 2A)

Simultaneously the area of beating in the presence of ECP was nearly 8-fold wider than

that without ECP The average area of beating was 48 plusmn 1047 per field (n = 24 different

fields field size = 08 mm2) with ECP versus 617 plusmn 123 (n = 24 different fields) without

ECP (Fig 2B and C) Thus ECP was judged to enhance cardiomyocyte differentiation in

both beating rate and area However ECP did not induce cardiomyocyte differentiation

without DMSO treatment

- 26 -

Enhanced expression of cardiomyocyte genes in P19CL6 cells after ECP treatment

The time course change from 4 to 12 days of cardiac marker gene expression in P19CL6

cells was evaluated by RT-PCR in the presence of ECP and DMSO (Fig 3A) GATA-4

expression was dramatically accelerated within 4 days of treatment with ECP and DMSO

when compared with that treated with only DMSO α-and β-MHC expression were also

accelerated within 8 days while α-MHC expression was upregulated within 12 days and

β-MHC expression still required more than 12 days for upregulation without ECP In

contrast to these genes Nkx25 expression was rather low However Nkx25 was

significantly upregulated within 12 days in the presence of ECP whereas it could not be

defected without ECP Further analyses on the expression of MLC GATA4 and α-MHC

were performed by real time qPCR and the effect of acceleration of cardiomyocyte

differentiation by ECP was confirmed (Fig 3B) P19CL6 cells at 12 days of treatment were

immunologically stained with antibodies against actinin actin and cardiac troponin I A

wider area of immunoreactivity which was indicative of enhanced expression in a greater

number of cells was observed in the presence of ECP than that without ECP (Fig 3C) Thus

the enhancement of cardiomyocyte differentiation by ECP was confirmed not only by

marker gene expression but also by the proteins expression

－

＋ 0

Fig 1 Morphological change of P19CL6 cells during cardiomyocyte differentiation

P19CL6 cells were treated with or without ECP in the presence of DMSO The cells were

ECP

Days 0 2 4 8 10 12

- 27 -

observed under microscope at indicated days The red frame shown the time that cells

started beating Magnification is times 20

Fig 2 Beating rate (A) and area (B) of cardiomyocytes differentiated from p19CL6 cells

treated for 12 days with or without ECP in the presence of DMSO (A) Beating rate was

calculated by beating frequency per minute of P19CL6 cells (A) (B) data represent mean plusmn

SD from triplicate of experiments Each asterisk shows the significance of Plt005 (C)

Typical area of beating cells (in white circle) counted in (B) The magnification is times 20

- 28 -

Fig 3 Induction of cardiac-specific genes (GATA4 Nkx25 MHC) assessed by RT-PCR

P19CL6 cells were treated with or without ECP in the presence of DMSO Total RNA was

isolated at indicated time points and subjected to RT-PCR

A B C

Fig 4 Expression of cardiac marker genes (GATA4 α-MHC MLC) assessed by real time

qPCR (A)(B)(C) P19CL6 cells were treated with or without ECP in the presence of DMSO

Total RNA was isolated at indicated time points and subjected to real time qPCR Each

result is represented as mean plusmn SD from triplicate of experiments

Fig 5 Immunostaining of cardiac specific proteins (Actinin and actin) P19CL6 cells were

treated with or without ECP in the presence of DMSO On 12 days the cells were

incubated with anti-actin and anti-actinin antibodies and then incubated with both Alexa

- 29 -

488-conjugated anti-mouse IgG antibody and Alexa Fluorreg 555-conjugated anti-rabbit IgG

secondary antibody Magnification is times 63 Scale Bars = 50 μm (C) Actinin (green) and

actin (red)

A B

Fig 6 Immunostaining of cardiac specific proteins (Cardiac troponin I and ANF) (A)

P19CL6 cells were treated with or without ECP in the presence of DMSO On 12 days the

cells were incubated with anti- cardiac troponin I actin antibodies and then incubated with

both Alexa 488-conjugated anti-mouse IgG antibody (B) The cells incubated as in (A) on

12 days the cells were incubated with anti- ANF and then incubated with Alexa

555-conjugated anti-mouse IgG antibody Magnification is times 63 Scale Bars = 50 μm

Discussion







ANF

ANF

- 30 -



access to a transplantation program to a few thousand patients a year [3 4] In this context

alternative approaches may include restoring heart function via induction of endogenous

regenerative processes and transferring progenitor cells to produce new myocardium

Cardiomyocytes are currently thought to be postmitotic cells that withdraw from the cell

cycle soon after birth Nevertheless mitoses in the myocyte population have been detected

in the human heart after myocardial infarction [5] Recent studies suggest that

cardiomyocytes may be continually replaced in the heart through processes involving





embryonic stem (ES) cells could be produced in large numbers differentiated to

cardiomyocytes and then used as a renewable source for cardiac cell therapy [8] One of the

major obstacles hindering the clinical use of ES cells-derived cardiomyocytes for cardiac

regenerative therapy is inefficient cardiac differentiation leading to insufficient amount of

cardiomyocytes [9] and the potential development of teratomas The most common

approach to solve this problem is to apply knowledge of developmental biology in

employing cardiogenesis-related factors for inducing stem cells cardiomyocyte


In the present study using P19CL6 cells we evaluated the potential of ECP as a

cardiomyocyte differentiation factor ECP upregulated the expression of cardiomyocyte

marker genes such as GATA4 α-MHC β-MHC Nkx25 and MLC in the presence of DMSO

together with the immunostaining for actinin actin and cardiac troponin I (Fig 5C) This is

the first study to report a function for ECP in cardiomyocyte differentiation from stem cells

in vitro We are now trying to develop ECP as cardiomyocyte differentiation factor which

could be used in the development of novel therapies for cardiac regeneration

- 31 -

Reference

1 P Hodges Heart failure epidemiologic update Crit Care Nurs Q 32 (2009) 24ndash32










Nadal-Ginard F Silvestri A Leri CA Beltrami P Anversa Evidence that human

cardiac myocytes divide after myocardial infarction N Engl J Med 344 (2001)

1750ndash1757


415 (2002) 240ndash243



refresh adult mammalian cardiomyocytes after injury Nat Med 13 (2007) 970ndash974







derived from human embryonic stem cells in pro-survival factors enhance function of

- 32 -

infarcted rat hearts Nat Biotechnol 251015-1024





Fukami S Shimazaki T Okano H Ogawa S Fukuda K 2005 Transient inhibition of

BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic

stem cells Nature Biotechnology 23897-897

13 Monzen K Hiroi Y Kudoh S Akazawa H Oka T Takimoto E Hayashi D Hosoda T

Kawabata M Miyazono K Ishii S Yazaki Y Nagai R Komuro I 2001 Smads

TAK1 and their common target ATF-2 play a critical role in cardiomyocyte

differentiation J Cell Biol 153687-698

14 Monzen K Shiojima I Hiroi Y Kudoh S Oka T Takimoto E Hayashi D Hosoda T

Habara-Ohkubo A Nakaoka T Fujita T Yazaki Y Komuro I 1999 Bone

morphogenetic proteins induce cardiomyocyte differentiation through the

mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors

CsxNkx-25 and GATA-4 Mol Cell Biol 197096-7105

15 Iwane M Kurokawa T Sasada R Seno M Nakagawa S Igarashi K 1987 Expression

of cDNA-encoding human basic fibroblast growth factor in Escherichia coli Biochem

Biophys Res Commun 146470-477

16 Maeda T Kitazoe M Tada H de Llorens R Salomon DS Ueda M Yamada H Seno

M 2002 Growth inhibition of mammalian cells by eosinophil cationic protein Eur J

Biochem 269307-316

17 Clement CA Kristensen SG Mollgard K Pazour GJ Yoder BK Larsen LA

Christensen ST 2009 The primary cilium coordinates early cardiogenesis and

hedgehog signaling in cardiomyocyte differentiation J Cell Sci 1223070-3082




- 33 -



19 Harada K Ogai A Takahashi T Kitakaze M Matsubara H Oh H 2008

Crossveinless-2 controls bone morphogenetic protein signaling during early

cardiomyocyte differentiation in P19 cells J Biol Chem 28326705-26713

20 Cohn JN Bristow MR Chien KR et al Report of the National Heart Lung And

Blood Institute Special Emphasis Panel On Heart Failure Research Circulation 1997

95766ndash770

21 Fedak PW Verma S Weisel RD et al Cardiac remodeling and failure From

molecules to man (Part I) Cardiovasc Pathol 2005 141ndash11

22 HABARA-OHKUBO A (1996) Differentiation of beating cardiac muscle cells from

a derivative of P19 embryonal carcinoma cells Cell StructFunct 21 101-110

23 Jackson KA Majka SM Wang H Pocius J Hartley CJ Majesky MW

Entman ML Michael LH Hirschi KK and Goodell MA (2001) Regeneration

of ischemic cardiac muscle and vascular endothelium by adult stem cells J Clin

Invest 107 1ndash8

24 Van der Heyden MA and Defize LH (2003) Twenty one years of P19 cells what

an embryonal carcinoma cell line taught us about cardiomyocyte differentiation

Car-diovascular Research 58 292-302

25 Anisimov SV Tarasov KV Riordon D Wobus AM and Boheler KR (2002)

SAGE identification of differ-entiation responsive genes in P19 embryonic cells

induced to form cardiomyocytes in vitro Mechanisms of Devel-opment 117 25-74

26 McBurney MW Jones-Villeneuve EM Edwards MK and Anderson PJ (1982)

Control of muscle and neuronal differentiation in a cultured embryonal carcinoma cell

line Nature 299 165-167

27 McBurney MW (1993) P19 embryonal carcinoma cells International Journal of

Developmental Biology 37 135-140

28 Skerjanc IS (1999) Cardiac and skeletal muscle development in P19 embryonal

carcinoma cells Trends Car-diovascular Medicine 9 139-143

- 34 -

29 Skerjanc IS Petropoulos H Ridgeway AG and Wil-ton S (1998) Myocyte

enhancer factor 2C and Nkx2-5 up-regulate each others expression and initiate

cardio-myogenesis in P19 cells Journal of Biological Chemistry 273 34904-34910

30 Wobus AM Kleppisch T Maltsev V and Hescheler J (1994) Cardiomyocyte like

cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by

functional expression of adrenoceptors and Ca2+ channels In Vitro Cellular

Development and Biology 30A 425-434

31 Rudnicki MA Jackowski G Saggin L and McBurney MW (1990) Actin and

myosin expression during devel-opment of cardiac muscle from cultured embryonal

car-cinoma cells Developmental Biology 138 348-358

32 Habara-Ohkubo A (1996) Differentiation of beating cardiac muscle cells from a

derivative of P19 embryonal carcinoma cells Cell Structure and Function 21

101-110

33 Van der Heyden MA van Kempen MJ Tsuji Y Rook MB Jongsma HJ and

Opthof T (2003) P19 embryonal carcinoma cells a suitable model system for cardiac

electrophysiological differentiation at the molecular and functional level

Cardiovascular Research 58 410-422

34 Eaton S Chatziandreou I Krywawych S Pen S Clayton PT and Hussain K

(2003) Short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency associated with

hyperinsulinism a novel glucose-fatty acid cycle Biochemical Society Transactions

31 1137-1139

35 Monzen K Hiroi Y Kudoh S Akazawa H Oka T Takimoto E Hayashi D

Hosoda T Kawabata M Miyazono K Ishii S Yazaki Y Nagai R and Komuro

I (2001) Smads TAK1 and their common target ATF-2 play a critical role in

cardiomyocyte differentiation Journal of Cell Biology 153 687-698

36 Paquin J Danalache BA Jankowski M McCann SM and Gutkowska J (2002)

Oxytocin induces differ-entiation of P19 embryonic stem cells to cardiomyocytes

Proceedings of the National Academy of Sciences USA 99 9550-9555

37 Peng CF Wei Y Levsky JM McDonald TV Childs G and Kitsis RN (2002)

- 35 -

Microarray analysis of global changes in gene expression during cardiac myocyte

dif-ferentiation Physioogical Genomics 9 145-155

38 Ridgeway AG Wilton S and Skerjanc IS (2000) Myocyte enhancer factor 2C

and myogenin up-regulate each others expression and induce the development of

skeletal muscle in P19 cells Journal of Biological Chemistry 275 41-46

39 Young DA Gavrilov S Pennington CJ Nuttall RK Edwards DR Kitsis RN

and Clark IM (2004) Expression of metalloproteinases and inhibitors in the

differentiation of P19CL6 cells into cardiac myocytes Biochemical and Biophysical

Research Communications 322 759-765

- 36 -

- 37 -

Chapter 2

Effect of eosinophil cationic protein (ECP) on activation

of FGF receptor 1 signaling during cardiomyocyte

differentiation

- 38 -

Abstract

I investigated the functional role of eosinophil cationic protein (ECP) in regulating

cardiomyogenesis using mouse P19CL6 embryonic carcinoma cells From previous chapter

ECP was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells by

enhancing the rate and area size of beating of cardiomyocyte and by facilitating the

expression of cardiomyocyte specific genes such as GATA-4 and α-MHC In this chapter I

detected the molecular mechanism of ECP on the cardiomyocyte differentiation of P19CL6

cells Since cardiomyocyte differentiation in vivo is considered to follow mesoderm

induction the induction of Brachyury a marker of mesoderm was assessed Brachyury

expression was found to be enhanced after the addition of ECP This enhancement was due

to the stimulation of ERK12 phosphorylation by ECP In this context treatment with

SU5402 an inhibitor of FGFR1 suppressed Brachyury expression phosphorylation of

ERK12 and cardiomyocyte differentiation induced by ECP We concluded that ECP might

induce mesoderm differentiation through FGF signaling pathway and enhance subsequent

cardiomyocyte differentiation in concert with DMSO in P19CL6 cells ECP may be a novel

factor for cardiomyocyte differentiation which should be very useful to prepare adequate

numbers of cardiomyocytes for therapeutic cell transplantation

- 39 -

Introduction

The heart is formed through multiple developmental steps which include the determination

of the cardiac field in the mesoderm differentiation of cardiovascular progenitor cells

differentiation of cardiac precursor cells and maturation of the heart Entry of cells into the

cardiac lineage is dependent upon appropriate external signals coupled to the expression of

a set of transcription factors that initiates the program for cardiac genes expression and

drives the morphogenic events involved in formation of the multichambered heart It is well

known that this process is complicated and so many signaling pathways included Research

in Mice Birds Amphibians Flies and Mammals as well as in various cell culture systems

has led to the identification of multiple transcription factors and extracellular growth factors

whose concerted actions specify the cardiac lineage in mesodermal progenitor cells The

earliest expressed transcription factors that initiate cardiac fate are the homeobox

transcription factor NKX25 and members of the GATA family of zinc finger transcription

factors GATA4 GATA5 and GATA6 Equally important roles in heart development have

been shown for members of the Tbx5 Tbx20 basic helix-loop-helix eHANDHAND1

(Heart and Neural crest Derivatives expressed-1)) and MADS (MCMI Agamous

Deficiens Serum response factor) domain (MEF2) families Extracellular signals that act

upstream of these factors have been primarily identified by their ability to induce cardiac

differentiation in non-cardiac mesoderm These signals belong to the BMP (Bone

Morphogenetic Protein) FGF (Fibroblast Growth Factor) and Wnt (Wingless-related

MMTV integration site) families of Growth Factors and also include secreted Wnt

antagonists such as Dkk1 (Dickkopf1) and Crescent [29-33]

The fibroblast growth factor (FGF) family is essential to normal heart development FGF2

and BMP signaling pathway play a crucial role in early Cardiomyogenesis Kawai T et al

reported FGF2 is required for the expression of Cardiac transcription factors and the

differentiation of mesoderm explants induced by BMP2 (Bone Morphogenetic Protein-2)

[34] FGF2 induces mesenchymal cell formation from precardiac mesoderm explants Other

- 40 -

members of the FGF family compensate for the lack of FGF2 expression in the embryo

such as FGF 4 and FGF 8 FGF-10 induced cardiomyocyte differentiation from ES cells

and iPS cells [28] In this chapter I detected the effect of ECP on activation of FGF

signaling pathway


Cell culture












Recombinant human ECP and recombinant human FGF-2 were expressed in bacteria and

prepared as described previously [22-24] SU5402 which is an FGF receptor (FGFR)

inhibitor was purchased from Calbiochem (San Diego CA) Anti-ERK and

anti-phospho-ERK12 mouse monoclonal antibodies were from New England Biolabs

(Beverly MA)All other reagents were of analytical grade and were purchased from Wako

or Sigma-Aldrich unless otherwise noted

Real time qPCR


- 41 -



Five micrograms of total RNA were then used to synthesize first-strand cDNA with

oligo-dT18 and 200 uunits SuperScript Ⅱ (Invitrogen CA) in a reaction volume of 20 μL

following the manufacturerrsquos instructions For quantitative analysis of gene expression

levels real time qPCR were performed and the data were normalized to GAPDH Primers

used for the real time qPCR were as following (forward and reverse) Wnt3a

5-AAGCAGGCTCTGGGCAGCTA-3 and 5-GACGGTGGTGCAGTTCCA-3

Brachyury 5-AGCTCTCCAACCTATGCGGACAAT-3 and











Western blotting

Cells grown on culture dishes were washed twice with ice-cold PBS and then suspended in

50 mM TrisndashHCl pH 74 containing 150 mM NaCl 1 mM EDTA 1 NP-40

supplemented with phosphatase inhibitors 10 mM sodium fluoride and 1 mM

phenylmethylsulfonyl fluoride 1 mM sodium orthovanadate and 10 mM sodium

pyrophosphate The cells were then disrupted by a sonicator (Ultras Homogenizer VP-53

TAITEC JP) with a microprobe setting at level 2 for 30 sec on ice and centrifuged at

13000g for 20 min at 4degC The supernatants were then carefully collected and stored at

-80degC until use Protein concentration was determined by BCA Protein Assay kit (Pierce

- 42 -

IL) and 20 microg of protein was subjected to 125 SDS polyacrylamide gel electrophoresis

and subsequently transferred onto PVDF membranes (Millipore MA) by semidry blotting

After being washed with Tris-buffered saline pH 76 containing 01 Tween 20 (TBST)

membranes were incubated for 1 h at 25 ˚C in TBST containing 5 BSA and the antigens

were probed with anti-ERK or anti-phospho-ERK12 antibodies diluted at 11000 at 4 ˚C

for 16 h After three washes with TBST membranes were probed with

horseradish-peroxidase-conjugated anti-rabbit antibody diluted at 12000 for 1 h at 25 ˚C

Following by three washes with TBST horseradish-peroxidase on the membrane was

reacted with the substrate kit (Western lightening plus-ECL Western blotting detection

reagents PerkinElmer US) and then detected by LSA-4000 (FUJIFILM Japan)


All experiments were replicated at least three times Results are described as means plusmn SD

To compare the differentiation in P19CL6 cells stimulated with ECP at different

concentration and durations One-way ANOVA followed by post hoc turkey analysis was

employed to assess the significance between the groups more than two Marker gene

expression beating rate beating area were analyzed by paired T-tests The statistical

software used for analysis was SPSS (version 170) P lt 005 was considered statistically

significant

Results

Effect of ECP on early cardiomyocyte differentiation in P19CL6 cells

During the early stages of cardiomyocyte differentiation P19CL6 cells express the

mesenndomerm genes Wnt-3a and Brachyury [25 26] The expression of these two genes

was therefore assessed in the presence of ECP (Fig 1) Expression of Wnt-3a and

Brachyury were transiently increased within 2 days after treatment with both ECP and

DMSO However expression of both genes was delayed by 2 days without ECP with

maximum expression at 4 days with only DMSO In addition the level of Wnt-3a and

- 43 -

Brachyury expression after 2 days of ECP treatment was significantly higher than

expression levels ascertained after 4 days of treatment with only DMSO This delay may

suggest that ECP accelerated the process of cardiomyocyte differentiation from

mesendoderm up to beating cardiomyocytes with respect to generating a greater number of

beating cardiomyocytes at earlier time

We therefore evaluated the effect of ECP on the early stages of cardiomyocyte

differentiation in P19CL6 cells First the expression profiles of GATA-4 and α-MHC genes

were assessed when ECP treatment was limited for the first 2 days or 4 days (Fig2) Both

genes were upregulated showing a similar pattern with those when treated with ECP and

DMSO whole through the entire period This suggests that ECP should engage a rapid

change in gene expression with the first 48 hours following treatment with DMSO

To determine concentration of ECP ECP at 10100 and 1000 ngmL in the presence of

DMSO was evaluated for its ability to stimulate cardiomyocyte differentiation of P19CL6

cells in the presence of 1 DMSO (Fig 3) Cardiomyocyte differentiation was monitored

by assessing the expression of GATA-4 and α-MHC genes by real time qPCR ECP at 1000

ngmL was found to maximally induce the expression of GATA4 and α-MHC at the earliest

time of differentiation on day 8(Fig 3) The effect of different concentration of ECP was

further assessed on the expression of Brachyury at the earlier stages of within a 48 h

window in the absence of 1 DMSO treatment Brachyury gene expression was

upregulated not only in a dose-dependent manner of ECP (Fig 4A) but also in a

time-dependent manner(Fig 4B) Maximum upregulation occurred at 24 h following

treatment with ECP at 1000 ngmL However ECP alone did not induce end stage

cardiomyocyte differentiation as reflected by beating cardiomyocyte Stimulation with ECP

at 1000 ngmL for only 24 h was able to significantly induce GATA-4 and α-MHC

expression in P19CL6 cells in the presence of DMSO (Fig 5) as well as a beating

phenotype after 8 days Therefore cardiomyocyte differentiation of P19CL6 cells can be

induced by 1 DMSO and enhanced by the stimulation with ECP at 1000ngmL following

a relatively short 24 h treatment

- 44 -

Stimulation of ERK12 phosphorylation by ECP

Considering the result that ECP could induce Brachyury expression in P19CL6 cells within

24h we hypothesized that ECP might induce mesendoderm formation leading to

subsequent cardiomyocyte differentiation Since Brachyury is a marker for mesendoderm

ERK12 phosphorylation has been described important for mesoderm induction [26] (Yao

et al 2003) Hence the time course change in the phosphorylation of ERK12 was assessed

in P19CL6 cells when stimulated with ECP (Fig 6) ECP upregulated the phosphorylation

of ERK12 after 5 minutes stimulation while DMSO alone did not show any effect on

ERK12 phosphorylation FGF2 also stimulated phosphorylation of ERK12 in a pattern

which was similar to ECP stimulation of ERK12 phosphorylation The effect of FGF2 on

enhancing ERK12 phosphorylation was delayed by the inclusion of DMSO

FGFR signaling pathway is involved in the stimulation of p-ERK by ECP

Brachyury expression is regulated by FGF through an ERK12-dependent signaling

pathway that is involved in mesoderm induction [27] To investigate the possible

involvement of an FGF receptor (FGFR) signaling pathway in ECP stimulation inhibition

of FGFR1 with SU5402 was evaluated SU5402 suppressed the phosphorylation of ERK12

that was induced by ECP at 5 minute (Fig 7B and C) In addition the expression of

Brachyury that was induced by ECP was suppressed in the presence of SU5402 (Fig 8)

These results suggest that ECP utilize an FGFR signaling pathway to initiate mesoderm

induction in P19CL6 cells Furthermore the protractive differentiation of P19CL6 cells into

cardiomyocytes was evaluated in the continuous presence of ECP and SU5402 for 2 days

by the expression of GATA-4 and α-MHC These two genes were downregulated by

SU5402 even in the presence of DMSO or DMSO and ECP (Fig 9) In the presence of

SU5402 for 2 or 4 days no beating cardiomyocyte phenotype appeared at day 8 and 12 in

the presence of DMSO even after ECP stimulation Collectively the induction of mesoderm

lineages through an FGFR1 signaling pathway is essentially dependent on DMSO and

enhanced by ECP Interestingly FGF2 unlike ECP did not induce cardiomyocyte

differentiation in P19CL6 cells either in the presence or absence of DMSO In contrast

- 45 -

Brachyury expression was induced additively on the effect of DMSO by ECP while FGF2

did not induce this additive effect (Fig 8)

Fig 1 Effect of ECP on Expression of genes associated with mesoderm in the presence of

DMSO (A)(B) Expression of Wnt3a and Brachyury genes P19CL6 cells were treated with

or without ECP in the presence of DMSO Total RNA was isolated at indicated time points

- 46 -

and subjected to real time qPCR Each result is represented as mean plusmn SD from triplicate of

experiments

Fig 2 The effects of different duration of stimulation by ECP on cardiomyocyte

differentiation of P19CL6 cells (A) P19CL6 cells were treated with ECP for the first 2 and

4 days in the presence of DMSO as shown in the schematic diagram(B)(C) P19CL6 cells

were treated with or without ECP in the presence of DMSO Total RNA was isolated at

indicated time points and subjected to real time qPCR The expression of GATA4 and

α-MHC genes was evaluated at indicated days Each result is represented as mean plusmn SD

from triplicate of experiments The statistic significance was assessed by one-way ANOVA

(see Material and Methods) Each asterisk shows the significance of Plt005

- 47 -

Fig 3 Cardiomyocyte differentiation at different concentration of ECP (A)(B) P19CL6

cells were treated with indicated concentration of ECP for first 2 days in the presence of

DMSO The expression of GATA4 and α-MHC genes was evaluated at indicated days

Each result is represented as mean plusmn SD from triplicate of experiments The statistic

significance was assessed by one-way ANOVA (see Material and Methods) Each asterisk

shows the significance of Plt005

- 48 -

Fig 4 ECP induced mesoderm marker gene Brachyury expression without DMSO (A)

Time course of Brachyury induction by ECP P19CL6 cells were treated with different

concentration of ECP At indicated time points Total RNA was isolated from the cells and

subjected to real time qPCR (B) Brachyury induction by ECP P19CL6 cells were treated

for 24 hours with different concentration of ECP The expression of Brachyury was

upregulated in a dose-dependent manner (A) (B) Each result is represented as mean plusmn SD



- 49 -

Fig 5 Cardiomyocyte differentiation of P19CL6 cells treated with ECP for one day (A)

Schematic diagram of the culturing conditions for P19CL6 cells with or without ECP for

one day in the presence of DMSO Total RNA was isolated at day 4 and day 8 (indicated by

asterisks) (B) Results from real time qPCR for the expression of cardiomyocyte marker

genes (GATA4 and α-MHC)

- 50 -

- 51 -

Fig 6 Effects of ECP FGF2 and DMSO on the phosphorylation of ERK12 in P19CL6

cells P19CL6 cells treated with ECP FGF2 and DMSO P19CL6 cells were analyzed by

western blot with anti-ERK antibody and anti-p-ERK12 antibody Each blot was

densitometrically quantified by Image J software In each panel the level of p-ERK12

after stimulated are presented as relative values against it at time 0 Total ERK12 was used

for normalization Each asterisk shows the significance of Plt005

- 52 -

Fig 7 Induction and phosphorylation of ERK12 by ECP is dependent on FGFR1 signaling

pathway (A) (B) Phosphorylation of ERK12 in P19CL6 cells by FGF2 ECP and its

inhibition by SU5402 (C) Phosphorylation of ERK12 in the P19CL6 cells by ECP in the

presence of DMSO All western blots were densitometrically quantified by Image J

software In each panel the level of p-ERK12 after stimulated are presented as relative

values against it in unstimulated cells Total ERK12 was used for normalization Each

asterisk shows the significance of Plt005

Fig 8 ECP induced Brachyury expression was suppressed by SU5402 in P19CL6 cells

P19CL6 cells were treated with DMSO ECP FGF and SU5402 for different combinations

Cells total RNA isolated at 24 hours and expression of Brachyury (a mesoderm marker)

was analyzed by real time qPCR The results are represented by mean plusmn SD from triplicate

of experiments Single-asterisk and double-asterisk show the significance of Plt005

- 53 -

Fig 9 FGFR inhibitor SU5402 suppressed cardiomyocyte differentiation in P19CL6 cells in

the presence of DMSO (A)(B) The P19CL6 cells were treated with inhibitor SU5402 for

the first 2 and 4 days in the presence of DMSOGATA4 and α-MHC by real time qPCR

Cells were collected at indicated time points and analyzed cardiac marker genes expression

by real time qPCR The expression of GATA4 and α-MHC genes was evaluated at indicated

days Each result is represented as mean plusmn SD from triplicate of experiments The statistic

significance was assessed by one-way ANOVA (see Material and Methods) asterisk show

the significance of Plt005

- 54 -

Fig 10 FGFR inhibitor SU5402 suppressed cardiomyocyte differentiation in P19CL6 cells

Cells were treated with or without ECP in the presence of DMSO (A)(B) (A) (B) Cells

were collected at indicated time points and analyzed cardiac marker genes expression by

real time qPCR The expression of GATA4 and α-MHC genes was evaluated at indicated




Discussion

The eosinophil granulocyte takes part in the bodyrsquos natural defense against invading

parasites bacterials and virus [1-3] as well as in other inflammatory diseases like allergic

asthma and gastrointestinal disorders [4] Once attracted to the site of inflammation the

eosinophil becomes activated and as a result of this eosinophil degranulated mainly

releases secretes 4 proteins These are the major basic protein (MBP) present in their cores

surrounded by a matrix built up of eosinophil peroxidise (EPO) the eosinophil protein

Xeosinophil derived neurotoxin (EPXEDN) and ECP But it was not confirmed until to

- 55 -

1975 it become clear ECP was of eosinophil origin Since the discovery of ECP in 1971

more than 3000 articles have been published about ECP focus on dealing with molecular

and genetic characteristics and functions of ECP and the majority describing the

distribution of ECP in tissues and body fluids in patients with diseases such as asthma and

other inflammatory diseases

The major challengeproblem of using ES cell-based cardiomyocytes replacement therapy

is the inefficiency of the conventional protocol to generate cardiomyocytes [5 6] It is

crucial for researchers to find novel inducers of cardiomyocyte differentiation and to clearly

understand the mechanism of differentiation ECP can upregulate transforming growth

factor alpha expression in human lung fibroblast cells [7] and can enhance stress fiber

formation in mouse fibroblasts [8] Simultaneously we demonstrated that ECP induced the

differentiation of cardiomyocytes in rat neonates as judged by a increase in both the beating

rate and the expression of cardiac muscle specific markers such as atrial natriuretic factor

[8] In the present study using P19CL6 cells we evaluated the potential of ECP as a



together with the immunostaining for actinin actin and cardiac troponin I (Fig 2C) In

the present study using P19CL6 cells we evaluated the potential of ECP as a

cardiomyocyte differentiation factor Actually ECP shortened the period of differentiation

by approximately at least 4 days until the appearance of a beating phenotype in P19CL6

cells while DMSO essentially primes differentiation in this system Early stages of

cardiomyocyte differentiation were reported to involve the transient upregulation of Wnt-3a

and Brachyury genes which were the typical markers of mesendoderm induction [9-11] As

shown in Fig 4 Brachyury expression was dependent on the dose of ECP in P19CL6 cells

The expression of Brachyury was maximally induced at 1000ngmL of ECP within 24 h

without DMSO

Wnt-3a induces mesoderm formation and cardiomyogenesis in embryonic stem cells [12]

Consistent with the result by Nakamula et al we observed temporal up-regulation of wnt3a

- 56 -

expression during DMSO induced the differentiation (Fig 3a) Wnt3a should activate

Wntβ-catenin signaling pathway presumably in autocrineparacrine manner which is

required for the cardiomyocyte differentiation in P19CL6 cells [13] We observed no

induction of Wnt3a expression by ECP alone (data not shown) In contrast ECP enhanced

and accelerated the expression of Wnt3a gene in presence of DMSO implicating that this

should be an indirect effect of ECP resulting from enhanced mesoderm differentiation

Extracellular signal-regulated kinase (ERK) was also shown to be necessary for mesoderm

differentiation in ES cells [14 15] In this study ECP was found to up-regulate the

phosphorylation of ERK12 in P19CL6 cells SU5402 a specific inhibitor of FGFR1 not

only downregulated phosphorylation of ERK12 but also inhibited Brachyury expression

abolishing the cardiomyocyte differentiation of P19CL6 cells following treatment with

ECP in the presence of DMSO ECP was found to stimulate the FGFR signaling pathway

However FGF2 did not enhance the cardiomyocyte differentiation in P19CL6 cells in the

presence of DMSO as ECP did FGF4 had the same negative result as FGF2 (data not

shown) FGF2 failed to enhance the Brachyury expression (Fig 8) and the phosphorylation

of ERK12 induced by FGF2 was delayed in the presence of DMSO (Fig 6) which differs

from the responses observed after ECP treatment These results might be due to the

binding of ECP to the FGFR which is different from FGF a region as conformation of the

FGF In this respect ECP has been reported to bind heparin and other glycosaminoglycans

like lectins through its RNase active site [16] FGF2 also binds to extracellular matrix

through syndecan 4 to exert some of its distinct biological activity [17] Binding of ECP to

the extracellular matrix components or cell surface GAG-containing proteins such as

syndecans and glypicans of P19CL6 cells should be explored to potentially explain the

modulation of FGFR signaling by ECP to enhance Brachyury expression The optimal

concentration of FGF2 to induce proliferation in fibroblasts is around 06 nM In this study

the optimal concentration of ECP was 1000ngmL which is equivalent to approximately 60

nM This 100-fold difference in biological activity may be due to different in binding

affinity to the FGFR by each protein ECP might be interacting with additional some

extracellular matrix proteins like syndecans which could alter the affinity of binding to the

- 57 -

FGFR Further studies are necessary to identify the effect of ECP on the activation of

FGFR signaling

SU5402 inhibited cardiomyocyte differentiation of P19CL6 cells in the presence of DMSO

Cardiomyocyte differentiation of P19CL6 cells appeared to depend on signaling through

the FGFR1 (Fig 9A) ECP induced ERK phosphorylation through FGFR1 while DMSO

alone did not affect the phosphorylation of ERK12 in P19CL6 cells (Fig 6)

Phosphatidylinositol 3-kinase (PI3K) plays an important role on the cardiomyocyte

differentiation of P19CL6 cells [18] Downstream intracellular signaling which is activated

by the FGFR could be due to both of PI3K-AKT and RAS-MAPK-ERK12 pathways [19]

ECP alone is not able to induce the cardiomyocyte differentiation in P19CL6 cells while

DMSO is essential for cardiomyocyte differentiation of P19CL6 cells The enhanced

phosphorylation of ERK12 that is induced by ECP through the FGFR1 should be closely

related with the accelerated cardiomyocyte differentiation because this phosphorylation was

not affected by DMSO while DMSO delayed the phosphorylation stimulated by FGF2

The expression of FGFR-1 in P19CL6 cells was confirmed by real time qPCR

(supplementary data) This might implicate our result that FGFR1 signaling pathway

triggered by ECP is involved in the accelerated differentiation of P19CL6 cell into

cardiomyocytes

This is the first study to report a function for ECP in cardiomyocyte differentiation from

stem cells in vitro We are now trying to develop ECP as cardiomyocyte differentiation

factor which could be used in the development of novel therapies for cardiac regeneration

- 58 -

Reference

1 McLaren DJ Peterson CG Venge P Schistosoma mansoni further studies of the

interaction between schistosomula and granulocyte-derived cationic proteins in vitro

Parasitol 1984 88491 503

2 Domachowske JB Dyer KD Adams AG Leto TL Rosenberg HF Eosinophil cationic

proteinRNase 3 is another RNase A-family ribonuclease with direct antiviral activity

(In process citation) Nucl Acids Res 1998 26 3358 63

3 Lehrer RI Szklarez D Barton A Ganz T Hamann KJ Gleich GJ Anti-bacterial

properties of eosinophil major basic protein (MBP) and eosinophil cationic protein

(ECP) J Immunol 1989 1424428 34

4 Venge P Eosinophils In Barnes P Rodger IA Thomson NC eds Asthma basic

Mechanisms and clinical management 3rd edn London Academic Press 1998141

57

5 Chan SS Chen JH Hwang SM Wang IJ Li HJ Lee RT Hsieh PC 2009 Salvianolic

acid B-vitamin C synergy in cardiac differentiation from embryonic stem cells

Biochem Biophys Res Commun 387723-728

6 Chan SS Li HJ Hsueh YC Lee DS Chen JH Hwang SM Chen CY Shih E Hsieh

PC 2010 Fibroblast growth factor-10 promotes cardiomyocyte differentiation from

embryonic and induced pluripotent stem cells PLoS One 5e14414

7 Chang HT Kao YL Wu CM Fan TC Lai YK Huang KL Chang YS Tsai JJ Chang

MD 2007 Signal peptide of eosinophil cationic protein upregulates transforming

growth factor-alpha expression in human cells J Cell Biochem 1001266-1275

8 Fukuda T Iwata M Kitazoe M Maeda T Salomon D Hirohata S Tanizawa K

Kuroda S Seno M 2009 Human eosinophil cationic protein enhances stress fiber

formation in Balbc 3T3 fibroblasts and differentiation of rat neonatal cardiomyocytes

Growth Factors 27228-236

- 59 -

9 Martin BL Kimelman D 2010 Brachyury establishes the embryonic mesodermal

progenitor niche Genes amp Development 242778-2783

10 Mauritz C Schwanke K Reppel M Neef S Katsirntaki K Maier LS Nguemo F

Menke S Haustein M Hescheler J Hasenfuss G Martin U 2008 Generation of

functional murine cardiac myocytes from induced pluripotent stem cells Circulation

118507-517

11 Mueller RL Huang C Ho RK 2010 Spatio-temporal regulation of Wnt and retinoic

acid signaling by tbx16spadetail during zebrafish mesoderm differentiation Bmc

Genomics 11492

12 Tran TH Wang X Browne C Zhang Y Schinke M Izumo S Burcin M 2009

Wnt3a-induced mesoderm formation and cardiomyogenesis in human embryonic

stem cells Stem Cells 271869-1878

13 Nakamura T Sano M Songyang Z Schneider MD 2003 A Wnt- and

beta-catenin-dependent pathway for mammalian cardiac myogenesis Proc Nat Acad

Sci USA 1005834-5839

14 Naito AT Akazawa H Takano H Minamino T Nagai T Aburatani H Komuro I 2005

Phosphatidylinositol 3-kinase-Akt pathway plays a critical role in early

cardiomyogenesis by regulating canonical Wnt signaling Circ Res 97144-151

15 Yao Y Li W Wu J Germann UA Su MS Kuida K Boucher DM 2003 Extracellular

signal-regulated kinase 2 is necessary for mesoderm differentiation Proc Natl Acad

Sci U S A 10012759-12764

16 Torrent M Odorizzi F Nogues MV Boix E 2010 Eosinophil cationic protein

aggregation identification of an N-terminus amyloid prone region

Biomacromolecules 111983-1990

17 Freund C Ward-van Oostwaard D Monshouwer-Kloots J van den Brink S van

Rooijen M Xu X

18 Giembycz MA Lindsay MA 1999 Pharmacology of the eosinophil Pharmacol Rev

51213-340

19 Gleich GJ 2000 Mechanisms of eosinophil-associated inflammation J Allergy Clin

- 60 -

Immunol 105651-663















Biochem 269307-316

24 Newton DL Nicholls PJ Rybak SM Youle RJ 1994 Expression and

Characterization of Recombinant Human Eosinophil-Derived Neurotoxin and

Eosinophil-Derived Neurotoxin-Anti-Transferrin Receptor Sfv J Biol Chem

26926739-26745

25 Jamali M Rogerson PJ Wilton S Skerjanc IS 2001 Nkx2-5 activity is essential for

cardiomyogenesis J Biol Chem 27642252-42258



Sci USA 1005834-5839

27 Schultemerker S Smith JC 1995 Mesoderm Formation in Response to Brachyury

Requires FGF Signaling Curr Biol 562-67



- 61 -


29 Schultheiss TM Burch JB Lassar AB A role for bone morphogenetic proteins in the

induction of cardiac myogenesis Genes Dev 1997 11 451ndash462

30 Alsan BH Schultheiss TM Regulation of avian cardiogenesis by Fgf8 signaling

Development 2002 129 1935ndash1943

31 Marvin MJ Di Rocco G Gardiner A Bush SM Lassar AB Inhibition of Wnt activity

induces heart formation from posterior mesoderm Genes Dev 2001 15 316ndash327

32 Schneider VA Mercola M Wnt antagonism initiates cardiogenesis in Xenopus laevis

Genes Dev 2001 15 304ndash315

33 Tzahor E Lassar AB Wnt signals from the neural tube block ectopic cardiogenesis


34 Kawai T Takahashi T Esaki M Ushikoshi H Nagano S Fujiwara H Kosai K 2004

Efficient cardiomyogenic differentiation of embryonic stem cell by fibroblast growth

factor 2 and bone morphogenetic protein 2 Circ J 68691ndash702

- 62 -

- 63 -

Chapter 3

Effect of ECP on the expression of genes correlated

with cardiomyocyte differentiation in P19CL6 cells

- 64 -

Abstract

We all know that cardiomyocyte differentiation is a complex process and so many signaling

pathways are included (such as FGF signaling Wnt signaling Eendothelin 1 (ET-1)

endothelin receptor A (ET-A) criptonodal signaling and so on) ECP effects are dependent

on DMSO on inducing cardiomyocyte differentiation in P19CL6 cells From chapter 3 we

have shown that ECP enhanced cardiomyocyte differentiation by stimulating FGFR1

signaling pathway In this chapter we checked effects of ECP on other signaling pathways

DMSO up-regulated Wnt3a expression and Wnt3a activated Wntβ-catenin signaling

pathway in autocrine manner This is required for the cardiomyocyte differentiation in

P19CL6 cells ECP alone can not induce Wnt3a expression However in the presence of

DMSO ECP accelerated Wnt3a suggested ECP stimulation have some positive effects on

the Wntβ-catenin signaling pathway in the presence of DMSO ECP activated PI3KAkt

signaling pathway It was well known that PI3KAkt signaling pathway is pivotal for

β-catenin (a down-stream molecule of Wnt) stabilization DMSO and ECP alone activated

this signaling respectively and ECP increased effects of DMSO on this signaling pathway

Through TOPFOP FLASH luciferase assay even if ECP did not induced Wnt3a expression

but it increased luciferase activity From above we get summary that ECP directly activated

PI3KAkt signaling and Wntβ-catenin pathway which was activated by DMSO

subsequently promote differentiation In addition we also confirmed that ECP also

enhanced EphA1 and ET-1 expression by DMSO both of signaling pathway correlated

with cardiomyocyte differentiation From above data it suggested that ECP through direct

and indirect manner activated signaling pathway subsequently influenced cardiomyocyte

differentiation

- 65 -

Introduction

It is well known that cardiomyocyte differentiation is complicated and so many signaling

pathways are included Proteins of the Wnt family are known regulators of

Cardiomyogenesis Wnts could bind to their transmembrane receptors Frizzled and

co-receptors LRP 56 on target cells to activate different signaling pathways Activation of

the canonical pathway leads to stabilization of β-catenin through inactivation of GSK3β

(Glycogen Synthase Kinase-3-β) β-catenin -dependent activation of TCF (T Cell Factor)

LEF (Lymphoid Enhancer Factor) transcription factors and induction of Wnt-responsive

genes This is canonical Wntβ-catenin signaling pathway In contrast Wnt11 signals

through a β-catenin -independent non-canonical pathway involving PKC (Protein

Kinase-C) and JNK (Jun-N-terminal kinase) Wnts induced Myogenic specification and

mammalian Cardiac myogenesis Wnt3a (Wingless-related MMTV integration site-3a)

upregulated early Cardiac marker genes before gastrulation in Mouse through stabilizing

β-catenin but inhibits heart formation during gastrulation [9] It suggests that

Wntβ-catenin signaling pathway plays a biphasic role in cardiomyocyte differentiation

Activation is required to commit mesenchymal cells to the cardiac lineage downregulation

of β-catenin is needed for cardiomyocyte differentiation at later stages Activation of

Wntβ-catenin signaling pathway during early EB formation enhances mouse ES cell

differentiation into cardiomyocytes and suppresses the differentiation into hematopoietic

and vascular cell lineages [10]

Eph was the first isolated member of what is currently the largest subfamily of RTKs

[11-23] This group is distinguished by a cysteine-rich region and two fibronectin type III

repeatsin the extracellular domain [12] Eph-subfamily kinases have been found in diverse

species including humans [14 17 19] mice [16 20 21] rats [13 15] zebrafish [23] and

chickens [12 16 18] Addictively EphA1 have a high expression level in embryonic stem

cells Features of their expression pattern suggest key functions during embryonic

development Jason D et al reported that Eph-related molecules have a potential role

during very early embryonic development [36] In addition to both of signaling above

- 66 -

NodalCripto signaling Endothelin 1 (ET-1) Endothelin 1 receptor A (ET-A) signaling

and Notch signaling have been investigated that these signaling are correlated with

cardiomyocyte differentiation during development stage In this report we are deciphering

the roles of ECP on these signaling pathways during cardiomyocyte differentiation in

P19CL6 cells


Cell culture


were cultured essentially as described previously[25 26] (Monzen et al 2001 Monzen et

al 1999) Briefly the cells were grown in 100-mm tissue culture dishes under adherent

conditions with α-minimum essential medium (α-MEM) (Invitrogen Tokyo Japan)

containing 10 fetal bovine serum (FBS) (Invitrogen) as growth medium in a 5 CO2

atmosphere at 37degC To induce cardiomyocyte differentiation under adherent conditions

P19CL6 cells were plated at a density of 5times105 in 60-mm tissue culture dishes in growth

medium Twenty four hours later the medium was replaced with growth medium

containing 1 DMSO (Nacalai tesque Kyoto Japan) as differentiation medium The

medium was changed to every 2 days and the cells were maintained under fresh conditions

until they started beating



[27 28] Epha1 siRNA and transfection reagent were purchased from Qiagen Anti-Akt was

prepared in our lab and anti-phospho-Akt mouse monoclonal antibodies were from New

England Biolabs (Beverly MA) All other reagents were of analytical grade and were

purchased from Wako or Sigma-Aldrich unless otherwise noted

Real time qPCR

- 67 -








used for the real time qPCR were as following (forward and reverse)

Wnt3a 5-AAGCAGGCTCTGGGCAGCTA-3 and 5-GACGGTGGTGCAGTTCCA-3

OCT4 5-TCTTTCCACCAGGCCCCCGGCTC-3 and

5-TGCGGGCGGACATGGGGAGATCC-3 FIk1

5-TAGCTGTCGCTCTGTGGTTCTG-3 and 5-GTCTTTCTGTTGTGCTGAGCTTGG-3

Nodal 5-TGCTGAAACGATACCAACCC-3 and

5-CCTGCCATTGTCCACATAAAGC-3 Frizzled 1

5-GCGACGTACTGAGCGGAGTG-3 and 5-TGATGGTGCGGATGCGGAAG-3

Frizzled 2 5-CTCAAGGTGCCGTCCTATCTCAG-3 and

5-GCAGCACAACACCGACCATG-3 Frizzled 4

5-GACAACTTTCACGCCGCTCATC-3 and

5-CCAGGCAAACCCAAATTCTCTCAG-3 Frizzled 8

5-GTTCAGTCATCAAGCAGCAAGGAG-3 and 5-AAGGCAGGCGACAACGACG-3

Frizzled 10 5-ATCGGCACTTCCTTCATCCTGTC-3 and

5-TCTTCCAGTAGTCCATGTTGAG-3 ET-1 5-ACTTCTGCCACCTGGACATC-3 and

5-ACTTTGGGCCCTGAGTTCTT-3 ET-A 5-ACCGTCTTGAACCTCTGTGC-3 and

5-AGCCACCAGTCCTTCACATC-3 Notch

5-AGATTGAGGCCGTGAAGAGTGAGC-3 and

5-CCACAAAGAACAGGAGCACGAAGG-3 EphA1

5-AACCTTATGCCAACTACACC-3 and 5-TTCCCCAAACTCTCCTTCTC-3

Luciferase assays

- 68 -

Plasmids TOPFlash contains TCF-LEF binding site upstream of the thymidine kinase

minimal promoter and luciferase cDNA Negative control counterpart FOPFlash has a

mutated TcfLef binding site (Upstate Biotechnology) P19CL6 cells were transfected by

using FuGENE 6 (Roche) with 02 μg of plasmid per 1times105 cells Luciferase activity was

determined by using a commercially available assay system (Promega) and was normalized

for transfection efficiency with pcDNA3-β-galactosidase

Western blot












membrane were incubated for 1 h at 25 ˚C in TBST containing 5 BSA and the antigens

were probed with anti-AKT or anti-phospho-Akt antibodies diluted at 11000 at 4 ˚C for 16

h After three washes with TBST membranes were probed with





Microrarray analysis

Expression of genes in P19CL6 cells was analyzed by microarray procedure Total RNA

- 69 -

preparation and analysis was performed as described previously [29 30] DNA microarray

was carrying 1428-oligonuclotide probes for human cell surface proteins cDNAs were

synthesized with Superescript Ⅱ reverse transcriptase (invitrogen) with oligo dT primers

Amino-allyl-dUTP was incorprated into cDNAs followed by coupling with Cy-3 dye

(Ambion TX) and were processed for hybridization at 55 for 15 hours The fluorescent

images for the hybridization were captured using FLA8000 scanner (Fuji film Japan) and

analyzed with GenePix Pro51 software (Axon Instruments CA) We found ECP or DMSO

only upregulated EphA1 expression in P19CL6 cells Moreover EphA1 expression was

enhanced in P19CL6 cells treated with ECP in the presence of DMSO Real time qPCR

further confirmed the results (Fig) Real time qPCR was performed with SYBR Green Real

time Master Mix (toyobo) in triplicates containing 5 ng of cDNA along with 400 nM

primers using LightCycler (Roche) The thermal cycling condition for real time qPCR was

as follows 95degC for 5 min followed by 40 cycles of denaturation at 95degC for 10 sec

annealing at 58degC depending on the melting temperature of each pair of primers for 10 sec

followed by extension at 72degC for 10s The following sets of primers were used for the

PCR reaction EphA1 5-TCACGCTGCCTCTGCTGTCT-3 and

5-GCCCACCAGTTTCCAGAAGCCT-3

EphA1 siRNA transfection

P19CL6 cells were plated at a density of 04times105 well in 24 well plates in growth medium

and incubated one hour in a 5 CO2 atmosphere at 37degC EphA1 siRNA and control

siRNA 75 ng were diluted with 100 μl OPTI-MEM culture medium with no FBS final

concentration is 10 nM and mix with 3μl HiperFect Transfection Reagent by vortexing

then incubated 10 minutes at room temperature The mixtures were then added dropwise

into the well with cells and siRNA concentrations were obtained to 10 nM in the medium

After 24 hours incubation the cells were induced differentiation in the presence of DMSO

with or without ECP Real time qPCR was performed to analysis the knockdown efficiency

of EphA1 and cardiomyocyte differentiation

- 70 -

Results

Effect of ECP on the Wntβ-catenin signaling pathway

Because canonical Wnt signaling pathway plays an essential role in cardiomyocyte

differentiation so in this chapter I first examined Wnt3a expression in P19CL6 cells

treated with different concentration of ECP I observed no induction of Wnt3a expression

by ECP alone (Fig 1) In contrast ECP enhanced and accelerated the expression of Wnt3a

gene in presence of DMSO Luciferase activity in P19CL6 cells tranfected with

TOPFLASH plasmid (Fig 2) which reflects the stabilization ofβ-catenin was elevated on

day 1 and 2 after stimulation of ECP withwithout DMSO And then we analyzed the Wnt

receptor genes (Frizzleds) expression ECP accelerated and enhanced frizzled 4 and 10 (Fig

3C and E) and enhanced Frizzled 1 (Fig 3A) expression in P19CL6 cells in the presence of

DMSO during cardiomyocyte differentiation However expression of frizzled 2 and 8 genes

has no significant change These results indicate that ECP activated Wnt signaling pathway

through directly enhanced stabilization of β -catenin and subsequently enhanced

cardiomyocyte differentiation

Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -

Fig 1 Effect of ECP on Wnt3a gene expression P19CL6 cells were treated with different

concentration of ECP for the first 2 days At indicated time points Total RNA was isolated

from the cells and subjected to real time qPCR ECP have no significant influence on the

Wnt3a gene expression

A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH

Fig 2 Effects of ECP on stabilization of β-catenin (A)(B) P19CL6 cells were transfected

with TOPFlash or FOPFlash plasmid in a concentration of 02 μg per 1times105 cells overnight

and then were stimulated with DMSO ECP for 1 day and 2 days respectively Luciferase

activity was shown as above the figures

A B

Frizzled-1

0

1

2

3

4

5

6

con 1 d 2 d 4 d 6 d 8 d 10 d 12 dTime course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45

con 1 d 2 d 4 d 6 d 8 d 10 d 12 dTime Course (days)

Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Fig 3 Effect of ECP on expression of Wnt receptor genes (Frizzleds ) in the presence of

DMSO (A) Frizzled 1 (B) Frizzled 2 (C) Frizzled 4 (D) Frizzled 8 (E) Frizzled 10

(A)-(E) P19CL6 cells were treated with or without ECP in the presence of DMSO Total

RNA was isolated at indicated time points and subjected to real time qPCR

Effects of ECP and DMSO on the phosphorylation of AKT in P19CL6 cells

- 73 -

Recently it has been reported that activation of PI3K is required for DMSO-induced

cardiomyocyte differentiation of P19CL6 cells Akt is one of the important downstream

molecules of PI3K Hence the time course change in the phosphorylation of Akt was

assessed in P19CL6 cells when stimulated with ECP (Fig 4) Phosphorylation of Akt was

upregulated in P19CL6 cells by ECP stimulation after 5 minutes in the presence of DMSO

or not

Fig 4 Effects of ECP and DMSO on the phosphorylation of Akt in P19CL6 cells P19CL6

cells treated with ECP and DMSO P19CL6 cells were analyzed by western blot with

anti-AKT antibody and anti-p-AKT antibody

Effect of ECP on Expression of Endothelin 1 (ET-1) and Endothelin receptor A genes

(ET-A) in the presence of DMSO

Papers have already published that ET-1 and ET-A are essential for cardiomyocyte

differentiation in P19 cells [35] Here we examined the expression of ET-1 and ET-A genes

by real time qPCR ECP enhanced both of genes expression in P19CL6 cells in the

presence of DMSO from 4 days (Fig 5) And then we used antagonist of ET-A (BQ123)

- 74 -

and ET-A and ET-B (another receptor of endothelins) (Bosentan) to treat P19CL6 cells in

the presence of DMSO then check the cardiomyocyte differentiation by real time qPCR

BQ123 and Bosentan suppressed the expression of cardiomyocyte marker gene in the

presence of DMSO (GATA4) (Fig 6) It suggested that ECP have a positive effect on

endothelins signaling

A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP

Fig 5 Effect of ECP on Expression of ET-1 and its receptor genes (ET-A ) in the presence

of DMSO (A) (B) P19CL6 cells were treated with or without ECP in the presence of

DMSO Total RNA was isolated at indicated time points and subjected to real time qPCR

A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8

Time course（day）

Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -

Fig 6 Effect of endothelins receptor A and receptor B antagonists (BQ123 and Bosentan)

on cardiomyocyte differentiation in P19CL6 cells in the presence of DMSO (A)(B) The

P19CL6 cells were treated with antagonist of endothelins receptor Bosentan (TRC) and

BQ-123(MERCK) in the presence of DMSO Total RNA was isolated at indicated time

points and analyzed cardiac marker genes expression by real time qPCR The expression of

GATA4 and α-MHC genes was evaluated at indicated days

Effect of ECP on NodalCripto Notch signaling pathway

Cripto an essential co-receptor of Nodal acts together with activin receptors to transmit the

Nodal signal via Smad2 and Smad3 [31-34] Previous studies have shown a requirement for

Nodal signaling in mesoderm differentiation in P19cells According to this point we

examined the expression of Nodal and its co-receptor Cripto genes Before 4 days both of

genes expression level was higher in P19CL6 cells treated with ECP in the presence of

DMSO over 4 days expression of two genes in the cells with ECP was significantly

downregulated however without ECP expression of two genes still maintained high level

from 4 days to eight days(Fig 7) Li et al reported that activation of Notch signaling in

undifferentiated P19CL6 cells promoted cardiac differentiation in this chapter ECP

accelerated Notch expression in the presence of DMSO (Fig 8)

A B

Fig 7 Effect of ECP on the expression of Nodal and its receptor (Cripto) gene in the

- 76 -

presence of DMSO (A) (B) P19CL6 cells were treated with or without ECP in the presence

of DMSO Total RNA was isolated at indicated time points and subjected to real time

qPCR

Fig 8 Effect of ECP on Expression of notch genes in the presence of DMSO (A) (B)


isolated at indicated time points and subjected to real time qPCR

Effect of ECP on EphA1 induction in P19CL6 cells with or without DMSO

Jason D et al investigated the potential role of Eph-related molecules during very early

embryonic development [36] In this chapter first of all we examined the Epha1

expression by using microarray at indicated days ECP upregulated the gene expression in

P19CL6 cells in the presence of DMSO or not To confirm this we checked the gene

expression by real time qPCR at indicated days (Fig 9) and got similar result with

microarray assays ECP treatment upregulated the expression of Epha1 gene Moreover

ECP enhanced Epha1 expression in the presence of DMSO compare to DMSO alone The

highest expression level of EphA1 was observed at one day These results indicated that

ECP activated EphA1 signaling pathway during very early embryonic development

- 77 -

Fig 9 Effect of ECP on the expression of EphA1 gene in the presence of DMSO P19CL6

cells were treated with or without ECP in the presence of DMSO Total RNA was isolated

at indicated time points and subjected to real time qPCR

Fig 10 EphA1 siRNA transfection P19CL6 cells were transfected with EphA1 siRNA

control means mock siRNA Total RNA was isolated at indicated time Knockdown

efficiency was examined by real time qPCR Results showed that EphA1 siRNA

suppressed EphA1 expression significantly

The roles of EphA1 signaling in the cardiomyocyte differentiation of P19CL6 cells

In order to investigate the roles of EphA1 signaling in the cardiomyocyte differentiation of

P19CL6 cells I knocked down the expression of EphA1 by siRNA Knockdown efficiency

- 78 -

was examined by real time qPCR EphA1 siRNA suppressed EphA1 expression

significantly (Fig 10) It is well known that cardiomyocyte differentiation of embryonic

stem cells is a very complicated procedure Cardiomyocyte differentiation of P19CL6 cells

underwent several stages denoted by the temporal expression of specific markers in the

presence of DMSO (Figure 11A and 11B) Undifferentiated P19CL6 cells (day 0) highly

expressed Oct4 which gradually diminished after differentiation Mesodermal cells

expressed Brachyury from day 1 to day 4 and cardiovascular progenitors the offspring of

mesodermal cells expressed Flk-1 from day 4 to day 8 Cardiac precursors started to appear

on day 4 indicated by the expression of Gata4 Cardiomyocytes as denoted by the

expression of the cardiac structural markers α-MHC expressed α-MHC appeared on day 12

However when the cells were treated with ECP the marker genes expression was

accelerated almost 4 days In order to examine effect of Epha1 signaling on cardiomyocyte

differentiation from P19CL6 cells we transfected P19CL6 with EphA1 siRNA after 1 day

induced P19CL6 cells with or without ECP in the presence of DMSO As shown in Fig 1

expression of these marker genes was suppressed (Fig 12) when the EphA1 gene was

knocked down even if mesoderm marker gene Brachyury expression These results

indicated that EphA1 signaling have a positive effect on mesoderm formation and then

influenced subsequent cardiomyocyte differentiation This is consistent with Jason D et al

reported that Eph-related molecules have a potential role during very early embryonic

development

A

- 79 -

B

Fig 11 Effects of ECP on different development stage of cardiomyocytes (A)The schemic

showed that marker genes expression at different stage of cardiomyocyte differentiation

(B) Marker genes expression was analyzed by real time qPCR P19CL6 cells were treated

with or without ECP in the presence of DMSO Total RNA was isolated at indicated time

points and subjected to real time qPCR

- 80 -

- 81 -

Fig 12 Effect of EphA1 siRNA on cardiomyocyte differentiation of P19CL6 cells

RT-qPCR results showed temporal expression patterns of these marker genes on days 1 2

4 and 8 during cardiomyocyte differentiation from P19CL6 cells Mock siRNA serve as

control

Discussion

Wntβ-catenin signaling plays pivotal roles in development of multiple tissues through

regulation of cell proliferation differentiation migration and gene expression [4]

Wntβ-catenin signaling pathway is focused on β-catenin stabilization In the inactivation of

canonical Wnt signaling pathway β-catenin complexed with APC and AXIN is

phosphorylated by glycogen synthase kinase 3β (GSK3β) in a degradation box which is

polyubiquitinated for proteasomal degradation [5] In the presence of Wnt ligands binding

to its receptor complex GSK3β is inhibited leading to stabilization of cytoplasmic

β-catenin Accumulated cytoplasmic β-catenin subsequently translocates to the nucleus and

initiates target gene transcription through T-cell factor (TCF)-lymphoid enhancer factor

(LEF) transcription factors [6 7] Wntβ-catenin signaling is critical for vertebrate cardiac

development This is same with our results Ablation of β-catenin in Isl1- expressing

cardiovascular progenitor cells disrupts multiple aspects of cardiogenesis resulting in

embryonic lethality at E13 [8] Other studies have shown a biphasic role of β-catenin in

cardiac specification The early activation of Wntβ-catenin signaling promotes cardiac

differentiation whereas the activation of Wntβ-catenin signaling at the later stage inhibits

heart formation [9] It has also been shown that the canonical Wnt signaling pathway

promotes commitment of P19CL6 cells into cardiac lineage at the early stage inhibiting

further differentiation into mature cardiomyocytes at the later stage [10] In this chapter we

examined the luciferase activity (Fig 2) which reflects the activity of canonical Wnt

signaling In P19CL6 cells luciferase activity was elevated on day 1 and 2 It implicated

that ECP had ability to activate Wnt signaling To confirm this we further examined the

- 82 -

expression of Frizzled genes Frizzled 1 4 10 three genes were upregulated in P19CL6

cells treated with ECP in the presence of DMSO (Fig 3) However after checked the

expression of Wnt3a by real time qPCR I found the gene expression was not upregulated in

P19CL6 cells treated with ECP These results suggested that activation of Wnt signaling is

required for the cardiomyocyte differentiation early event ECP activated Wnt signaling

through enhancing stabilization of β-catenin

Many studies have indicated that PI3KAkt signaling is involved in the differentiation of

various kinds of cells such as myoblasts[1] osteoblasts[2] and adipocytes[3] PI3K is

critically involved in cardiomyocyte differentiation in vitro It was reported that activation

of this signaling was required for cardiomyocyte differentiation of P19CL6 cells [10]

Activation of PI3KAkt pathway increased the content of cytoplasmic and nuclear β-catenin

and the activity of canonical Wnt pathway through increased GSK-3β phosphorylation So

in this study we examined phosphorylation of Akt which is important molecule

downstream of PI3K DMSO and ECP upregulated phosphorylation of Akt in P19CL6 cells

at 30 minutes This is consistent with Atsuhiko T et al reported that activation of

phosphatidylinositol 3-kinase (PI3K) plays a critical role in the early stage of

cardiomyocyte differentiation of P19CL6 cells [10]

Juan Carlos Monge et al reported that ET-1 and its receptor ET-A play critical role in

cardiomyocyte differentiation in P19 cells [35] In this chapter we detected both of genes

expression in P19CL6 cells with or without ECP in the presence of DMSO ECP enhanced

expression of two genes after 4 days compared to DMSO only It suggested that ECP had

positive effects on ET-1 and ET-A signaling and then enhanced cardiomyocyte

differentiation This is consistent with Juan Carlos Monge et al reported In addition we

used antagonist of ET-A to treat P19CL6 cells in the presence of DMSO GATA4

expression was downregulated And then we checked other genes expression correlated

with cardiomyocyte differentiation such as Nodal and its co-receptor Cripto genes

expression Notch expression ECP accelerated these genes expression Previous studies

have shown a requirement for Nodal Cripto signaling in mesoderm differentiation in P19

- 83 -

cells it implicated that ECP accelerated the mesoderm formation This is similar with our

result in chapter 2




species including zebrafish [23] chickens [12 16 18] mice [16 20 21] rats [13 15] and

humans [14 17 19] Additionally EphA1 have a high expression level in embryonic stem



during very early embryonic development [36] In this chapter I found that ECP enhanced

the expression of EphA1 in P19CL6 cells treated with ECP ECP with DMSO and DMSO

alone by microarray assay To further confirm this I used real time qPCR to examine again

I got the same results (Fig 9) as microarray assayed Furthermore I used siRNA of EphA1

to treated P19CL6 cells with or without ECP in the presence of DMSO checked the temple

profile marker genes expression during cardiomyocyte differentiation Knockdown of

EphA1 expression of stem cells marker gene OCT4 keep a high level In contract

Brachyury gene an early mesoderm marker gene expression was suppressed subsequently

influenced cardiomyocyte differentiation (Fig 11) These results were similar with Jason D

et al reported that Eph-related molecules have a potential role during very early embryonic

development [36]

From above data we summarize that ECP enhanced cardiomyocyte differentiation induced

by DMSO at early stage through initiating mesoderm differentiation by influencing several

signaling pathwaysuch as FGF Wnt PI3K EphA1

- 84 -

Reference

1 Jiang BH Aoki M Zheng JZ Li J Vogt PK Myogenic signaling of

phosphatidylinositol 3-kinase requires the serine-threonine kinase Aktprotein kinase

B Proc Natl Acad Sci U S A 1999 96 2077ndash2081

2 Ghosh-Choudhury N Abboud SL Nishimura R Celeste A Mahimainathan L

Choudhury GG Requirement of BMP-2-induced phosphatidylinositol 3-kinase and

Akt serinethreonine kinase in osteoblast differentiation and Smad-dependent BMP-2

gene transcription J Biol Chem 2002 277 33361ndash33368Epub 2002 Jun 25

3 Magun R Burgering BM Coffer PJ Pardasani D Lin Y Chabot J Sorisky A

Expression of a constitutively activated form of protein kinase B (c-Akt) in 3T3ndashL1

preadipose cells causes spontaneous differentiation Endocrinology 1996 137

3590ndash3593

4 Logan CY Nusse R 2004 The Wnt signaling pathway in development and disease

Annu Rev Cell Dev Biol 20 781ndash810

5 Katoh M Katoh M 2007 WNT signaling pathway and stem cell signaling network

Clin Cancer Res 13 4042ndash4045

6 Nakamura T Sano M Songyang Z Schneider MD 2003 A Wnt- and beta

-catenin-dependent pathway for mammalian cardiac myogenesis Proc Natl Acad Sci

USA 100 5834ndash5839

7 Deb A Davis BH Guo J Ni A Huang J Zhang Z Mu H Dzau VJ 2008 SFRP2

regulates cardiomyogenic differentiation by inhibiting a positive transcriptional

autofeedback loop of Wnt3a Stem Cells 26 35ndash44

8 Lin L Cui L Zhou W Dufort D Zhang X Cai CL Bu L Yang L Martin J Kemler R

Rosenfeld MG Chen J Evans SM 2007 Beta-catenin directly regulates Islet1

expression in cardiovascular progenitors and is required for multiple aspects of

cardiogenesis Proc Natl Acad Sci USA 104 9313ndash9318

9 Ueno S Weidinger G Osugi T Kohn AD Golob JL Pabon L Reinecke H Moon RT

- 85 -

Murry CE 2007 Biphasic role for Wntbeta-catenin signaling in cardiac specification

in zebrafish and embryonic stem cells Proc Natl Acad Sci USA 104 9685ndash9690



cardiomyogenesis by regulating canonical Wnt signaling Circ Res 97 144ndash151

11 Hirai H Maru Y Hagiwara K Nishida J amp Takaku F 1987 A novel

putativetyrosine kinase receptor encoded by the eph gene Science 238 1717-1720

12 Pasquale E B 1991 Identification of chicken embryo kinase 5 a developmentally

regulated receptor-type tyrosine kinase of the Eph family Cell Regul 2 523-534

13 Letwin K Yee S P amp Pawson T 1988 Novel protein-tyrosine kinase cDNAs

related to fpsfes and eph cloned using anti-phosphotyrosine antibody Oncogene 3

621-627

14 Lindberg R A amp Hunter T 1990 cDNA cloning and characterization of eck an

epithelial cell receptor protein-tyrosine kinase in the ephelk family of protein kinases

Mol Cell Biol 10 6316-6324

15 Chan J amp Watt V M 1991 eek and erk new members of the eph subclass of

receptor protein-tyrosine kinases Oncogene 6 1057-1061

16 Sajjadi F G Pasquale E B amp Subramani S 1991 Identification of a new

eph-related receptor tyrosine kinase gene from mouse and chicken that is

developmentally regulated and encodes at least two forms of the receptor New Biol

3769-3778

17 Boyd A W Ward L D Wicks I P Simpson R J Salvaris E Wilks A Welch

K Loudovaris M Rockman S ampBusmanis I 1992 Isolation and characterization

of a novel receptor-type protein tyrosine kinase (hek) from a human pre-B cell line J

Biol Chem 267 3262-3267

18 Wicks I P Wilkinson D Salvaris E amp Boyd A W 1992 Molecular cloning of

HEK the gene encoding a receptor tyrosine kinase expressed by human lymphoid

tumor cell lines Proc Natl Acad Sci USA 89 1611-1615

19 Sajjadi F G amp Pasquale E B 1993 Five novel avian Eph-related tyrosine kinases

- 86 -

are differentially expressed Oncogene 8 1807-1813

20 Bohme B Holtrich U Wolf G Luzius H Grzeschik K H Strebhardt K amp

Rubsamen W H 1993 PCR mediated detection of a new human

receptor-tyrosine-kinase HEK 2 Oncogene 8 2857-2862

21 Maisonpierre P C Barrezueta N X amp Yancopoulos G D 1993 Ehk-1 and Ehk-2

two novel members of the Eph receptor-like tyrosine kinase family with distinctive

structures and neuronal expression Oncogene 8 3277-3288

22 Henkemeyer M Marengere L E McGlade J Olivier J P Conlon R A

Holmyard D P Letwin K amp Pawson T 1994 Immunolocalization of the Nuk

receptor tyrosine kinase suggests roles in segmental patterning of the brain and

axonogenesis Oncogene9 1001-1014

23 Xu Q Holder N Patient R amp Wilson S W 1994 Spatially regulated expression

of three receptor tyrosine kinase genes during gastrulation in the zebrafish

Development Cambridge UK 120 287-299

24 J D Lickliter F M Smith J E Olsson K L Mackwell A W Boyd 1996 Embryonic

stem cells express multiple Eph-subfamily receptor tyrosine kinases Proc Natl Acad

Sci U S A January 9 93(1) 145ndash150













- 87 -



Biochem 269307-316

29 Tuoya Hirayama K Nagaoka T Yu D Fukuda T Tada H Yamada H Seno M 2005

Identification of cell surface marker candidates on SV-T2 cells using DNA microarray

on DLC-coated glass Biochem Biophys Res Commun Aug 19 334(1)263-8

30 Samah Abou-Sharieha Yuh Sugii Tuoya Dongwei Yu Ling Chen Heizou Tokutaka

and Masaharu Seno 2009 Identification of TM9SF2 as a candidate of the cell surface

marker common to breast carcinoma cells Clin Oncol Cancer Res (2009) 6 1-9

31 Reissmann E H Jornvall A Blokzijl O Andersson C Chang G Minchiotti MG

Persico CF Ibanez and AH Brivanlou 2001 The orphan receptor ALK7 and the

Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate

development Genes Dev 152010ndash2022

32 Gritsman K J Zhang S Cheng E Heckscher WS Talbot and AF Schier 1999

The EGF-CFC protein one-eyed pinhead is essential for nodal signaling Cell

97121ndash132

33 Yeo C and M Whitman 2001 Nodal signals to Smads through Cripto-dependent

and Cripto-independent mechanisms Mol Cell 7949ndash957

34 Yan YT JJ Liu Y Luo C E RS Haltiwanger C Abate-Shen and MM Shen

2002 Dual roles of Cripto as a ligand and coreceptor in the nodal signaling pathway

Mol Cell Biol 224439ndash4449

35 Juan Carlos Monge Duncan J Stewart Peter Cernacek Differentiation of Embryonal

Carcinoma Cells to a Neural or Cardiomyocyte Lineage Is Associated with Selective

Expression of Endothelin Receptors J Biol Chem 1995 270 15385-15390

36 J D Lickliter F M Smith J E Olsson K L Mackwell A W Boyd Embryonic stem

cells express multiple Eph-subfamily receptor tyrosine kinases Proc Natl Acad Sci U

S A 1996 93(1) 145ndash150

- 88 -

Conclusions

In this thesis the role of ECP on the cardiomyocyte differentiation was investigated in

mouse P19CL6 embryonic carcinoma cells I got conclusions as follows

1 ECP was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells

This is the first to report the function of ECP on cardiomyocyte differentiation from

stem cells in vitro

2 Since cardiomyocyte differentiation in vivo is considered to follow mesoderm induction

the induction of Brachyury a marker gene of mesoderm was assessed ECP enhanced

Brachyury expression

3 ECP induced mesoderm differentiation by stimulating FGF signaling pathway and

enhanced subsequent cardiomyocyte differentiation in concert with DMSO in P19CL6

cells

4 ECP alone can not induce Wnt3a expression However in the presence of DMSO ECP

accelerated Wnt3a expression increased TOPFLASH luciferase activity upregulated

some co-receptor frizzleds genes expression It suggested ECP stimulation have some

positive effects on the Wntβ-catenin signaling pathway in the presence of DMSO

5 ECP activated PI3KAkt signaling pathway through upregulating phosphorylation of

Akt

6 ECP enhanced EphA1 expression Aactivation of EphA1 signaling is required for the

cardiomyocyte differentiation in the P19CL6 cells

- 89 -

Acknowledgements

Completion of this doctoral dissertation was possible with the support of several people I

would like to express my sincere gratitude to all of them First of all I am extremely

grateful to my research guide Professor Masaharu Seno in Laboratory of Bioengineering

Graduate School of Nature Science and Technology Okayama University for his valuable

guidance discussion with inordinate patience reviewing and correction of the manuscript

and consistent encouragement I received throughout the research work I consider it as a

great opportunity to do my doctoral program under his guidance and to learn from his

research expertise The successful accomplishment of this thesis is mainly attributed to his

selfless contribution and excellent guidance

I am grateful to Dr Hiroshi Murakami for his guidance in statistical analysis and discussion

with inordinate patience I am also grateful to DrTakayuki Kudoh for his support and

motivation

I am deeply grateful to Dr Akifumi Mizutani for valuable advice kind technical assistance

reviewing and correction of the manuscript continuing encouragement and motivation I

am also grateful to Dr Tomonari Kasai for his kind helpful suggestion kind assistant and

humorous words

I would like to extend my sincere gratitude to the former post doc Dr Ling Chen and

Takayuki Fukuda for their support and motivation and contribution to the experiment

I would also like to extend my gratefulness to Dr Masashi Okada Dr Shuichi Matsuda Dr

Sreeja C Sekhar Dr Ting Yan and all the members of the laboratory for their kindly

- 90 -

discussion friendliness and encouragement I am also grateful to friends in Japan for their

encouragement warmth and friendly entertainments

I owe a lot to my parents who endured with extraordinary missing encouraged and helped

me at every stage of my personal and academic life and longed to see this achievement

come true

I am very much indebted to my wife who supported me in every possible way to see the

completion of this work

The financial support of the Japanese government is gratefully acknowledged

Guoliang Jin

Okayama University

September 2012

- 2 -

- 3 -

CONTENTS











- 4 -

Introduction















- 5 -

























- 6 -








Complex







- 7 -



code 1DYT














- 8 -


























Arg-34 [31]




- 9 -

















(RSV-B) [34]













- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 3 -

CONTENTS











- 4 -

Introduction















- 5 -

























- 6 -








Complex







- 7 -



code 1DYT














- 8 -


























Arg-34 [31]




- 9 -

















(RSV-B) [34]













- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 4 -

Introduction















- 5 -

























- 6 -








Complex







- 7 -



code 1DYT














- 8 -


























Arg-34 [31]




- 9 -

















(RSV-B) [34]













- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 5 -

























- 6 -








Complex







- 7 -



code 1DYT














- 8 -


























Arg-34 [31]




- 9 -

















(RSV-B) [34]













- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 6 -








Complex







- 7 -



code 1DYT














- 8 -


























Arg-34 [31]




- 9 -

















(RSV-B) [34]













- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 7 -



code 1DYT














- 8 -


























Arg-34 [31]




- 9 -

















(RSV-B) [34]













- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 8 -


























Arg-34 [31]




- 9 -

















(RSV-B) [34]













- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 9 -

















(RSV-B) [34]













- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 10 -


























[43]




- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 11 -





























- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 12 -

























- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 13 -

4 References








163-176

















p 45-50

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 14 -



233-240



S235-S235













S247-S247



Immunology 1995 95(1) p 277-277



p 511-511





- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 15 -





S8-S8



18-23



2012 24(4) p 393-397














1986 139(3) p 1239-1242





- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

- 16 -







Neurology 1953 12(4) p 349-362





1998 26(14) p 3358-3363








Chemistry 1995 270(14) p 7876-7881



Biochemistry 2003 42(22) p 6636-6644







- 17 -


1983-1990






179(1) p 1-9







56(4) p 353-358





Factors 2009 27(4) p 228-236


24ndash32








- 18 -





(2001) 1750ndash1757


415 (2002) 240ndash243




970ndash974
















- 19 -

Chapter 1




- 20 -

Abstract













- 21 -

Introduction



















on this research








- 22 -















Cell culture












- 23 -





Sigma-Aldrich
























- 24 -





























- 25 -







Results



















- 26 -


















－

＋ 0



ECP

Days 0 2 4 8 10 12

- 27 -








- 28 -




A B C








- 29 -



actin (red)

A B







Discussion







ANF

ANF

- 30 -





























- 31 -

Reference













1750ndash1757


415 (2002) 240ndash243











- 32 -























Biochem 269307-316







- 33 -








95766ndash770








Invest 107 1ndash8














- 34 -













101-110








31 1137-1139









- 35 -










- 36 -

- 37 -

Chapter 2



differentiation

- 38 -

Abstract

















- 39 -

Introduction



























- 40 -




signaling pathway


Cell culture


















Real time qPCR


- 41 -




















Western blotting









- 42 -


















significant

Results








- 43 -





























- 44 -





























- 45 -






- 46 -


experiments









- 47 -







- 48 -









- 49 -






- 50 -

- 51 -







- 52 -













- 53 -









- 54 -








Discussion








- 55 -


























without DMSO



- 56 -






























- 57 -


FGFR signaling
















cardiomyocytes




- 58 -

Reference



Parasitol 1984 88491 503






(ECP) J Immunol 1989 1424428 34



57














- 59 -






118507-517



Genomics 11492






Sci USA 1005834-5839






Sci U S A 10012759-12764





Rooijen M Xu X


51213-340


- 60 -

Immunol 105651-663















Biochem 269307-316




26926739-26745





Sci USA 1005834-5839





- 61 -















- 62 -

- 63 -

Chapter 3



- 64 -

Abstract






















differentiation

- 65 -

Introduction




























- 66 -





P19CL6 cells


Cell culture


















Real time qPCR

- 67 -




























Luciferase assays

- 68 -







Western blot





















- 69 -




























- 70 -

Results















Wnt3a

0

02

04

06

08

1

12

14

0 3 6 12 24 48

hours

Relative Expression

ECP 0ngml

ECP10ngml

ECP100ngml

ECP1000ngml

- 71 -





A B

0

05

1

15

2

25

3

35

con DMSO DMSO-ECP

ECP

Rel

ativ

e lu

cife

rase

act

ivit

y

TOPFLASHFOPFLASH

0

50

100

150

200

250

300

con DMSO DMSO-ECP

ECP

Rel

ativ

e L

ucif

eras

e ac

tivi

ty

TOPFLASHFOPFLASH





A B

Frizzled-1

0

1

2

3

4

5

6


Relative expression

DMSO

DMSO-ECP

Frizzled-2

0

05

1

15

2

25

3

35

4

45


Relative expression

DMSO

DMSO-ECP

- 72 -

C D

Frizzled-4

0

5

10

15

20

25

30

35

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP

Frizzled-8

0

2

4

6

8

10

12

14


Relative expression

DMSO

DMSO-ECP

E

Frizzled-10

0

10

20

30

40

50

60

con 1 d 2 d 4 d 6 d 8 d 10 d 12 d

Time course (days)

Relative expression

DMSO

DMSO-ECP






- 73 -






or not










- 74 -






A B

ET-1

0

20

40

60

80

100

120

0 1 2 4 6 8 10 12

Time Course (day)

Rel

ativ

e ex

pres

sion

DMSO

DMSO-ECP

ET-A

0

50

100

150

200

250

300

0 1 2 4 6 8 10 12

Time Course (day)

Relative expression

DMSO

DMSO-ECP




A B

GATA4

0

10

20

30

40

50

60

70

0 4 8

Time Course (day)

Rela

tive

exp

ress

ion

DMSO-BQ123

DMSO-BS

DMSO

MLC2

0

5

10

15

20

25

0 4 8


Rela

tive

exp

ress

ion

DMSO

DMSO-BQ123

DMSO-BS

- 75 -


















A B


- 76 -



qPCR














- 77 -











- 78 -




















development

A

- 79 -

B






- 80 -

- 81 -




control

Discussion






















- 82 -





























- 83 -


result in chapter 2


















development [36]




- 84 -

Reference











3590ndash3593







USA 100 5834ndash5839









- 85 -












621-627









3769-3778




Biol Chem 267 3262-3267





- 86 -






























- 87 -



Biochem 269307-316













97121ndash132











S A 1996 93(1) 145ndash150

- 88 -

Conclusions





stem cells in vitro






cells






Akt



- 89 -

Acknowledgements












motivation




humorous words





- 90 -





come true




Guoliang Jin

Okayama University

September 2012

Date post:	03-Feb-2022
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