Molecular and Functional Characterization of Uniform-Sized
Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic
and Induced Pluripotent Stem Cells

a 2014

Molecular and Functional Characterization of Uniform-Sized Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells

PEŠL, Martin, Ivana AĆIMOVIĆ, Jan PŘIBYL, Renata HÉŽOVÁ, Aleksandra VILOTIĆ et. al.

Základní údaje

Originální název

Molecular and Functional Characterization of Uniform-Sized Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells

Autoři

Vydání

58th Annual Meeting of the Biophysical-Society, 2014

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

Genetika a molekulární biologie

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 3.972

Organizační jednotka

Lékařská fakulta

ISSN

DOI

http://dx.doi.org/10.1016/j.bpj.2013.11.3135

UT WoS

000337000403197

Klíčová slova česky

human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium

Klíčová slova anglicky

human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium

Změněno: 8. 7. 2015 11:50, Ing. Mgr. Věra Pospíšilíková

Anotace

V originále

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are able to differentiate into functional cardiomyocytes (CMs). Traditional suspension method for 3D embryoid bodies (EBs) formation results in heterogeneous EB population. Here, we hypothesized that forming homogenous EB in size and shape allows studying the mechano-biological properties of spontaneously beating clusters containing CMs. Using a defined number of single cells in AggreWell plate, we formed homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors. We checked the expression of cardiac markers by qRT-PCR, immunocytochemistry and western blotting in both beating EBs and enzymatically-dissociated CMs and found increased expression of MYH6, MYH7 and RYR2 genes as well as sarcomeric pattern for cardiac troponin T and alpha-actinin. Using atomic force microscopy-based technique, we measured the beating properties of hESC-EBs and hiPSC-EBs and found a slower beat rate in hESC-CMs when compared to hiPSC-CMs (51 ± 5 vs 74 ± 7 bpm) and similar contraction force (31 ± 7 vs 39 ± 9 nN). Both cell types respond upon stimulation or inhibition of beta-adrenergic pathway and caffeine. Using Ca2+ imaging we also demonstrated the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum and evaluated the contribution of IP3R. Thus, upon xestospongin C and histamine (IP3R modulators), spontaneous Ca2+ transients are maintained confirming the role of RyR2. Using the patch-clamp technique, we evaluated the cardiac population constituting the EBs and found that more than 75% of excitable cells exhibit atrial-like action potentials. Our results indicated that the mechano-biological properties of homogenous beating EBs can be investigated. They also indicated that spontaneous beating EBs contain mostly functional and organized atrial CMs.

Návaznosti

ED1.1.00/02.0068, projekt VaV

Název: CEITEC - central european institute of technology

GA13-19910S, projekt VaV

Název: Studium dilatační kardiomyopatie spojené s Duchenovou svalovou dystrofií na modelové tkáni tvořené kardiomyocyty derivovanými z iPS buněk

Investor: Grantová agentura ČR, Studium dilatační kardiomyopatie spojené s Duchenovou svalovou dystrofií na modelové tkáni tvořené kardiomyocyty derivovanými z iPS buněk

MSM0021622430, záměr

Název: Funkční a molekulární charakteristiky nádorových a normálních kmenových buněk - identifikace cílů pro nová terapeutika a terapeutické strategie

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Funkční a molekulární charakteristiky nádorových a normálních kmenových buněk - identifikace cílů pro nová terapeutika a terapeutické strategie

7AMB13FR011, projekt VaV

Název: Přeprogramování somatických buněk darovaných pacienty s dědičnou Duchennovou svalovou dystrofií do kardiomyocytů - nahlédnutí do molekulární podstaty patologických dějů u dilatační kardiomyopatie nemocných DMD (Akronym: DUCHENSTEM)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Přeprogramování somatických buněk darovaných pacienty s dědičnou Duchennovou svalovou dystrofií do kardiomyocytů - nahlédnutí do molekulární podstaty patologických dějů u dilatační kardiomyopatie nemocných DMD.

Citovat

PEŠL, Martin, Ivana AĆIMOVIĆ, Jan PŘIBYL, Renata HÉŽOVÁ, Aleksandra VILOTIĆ, Franck AIMOND, Jeremy FAUCONNIER, Jan VRBSKÝ, Peter KRUŽLIAK, Petr SKLÁDAL, Tomáš KÁRA, Vladimír ROTREKL, Alain LACAMPAGNE, Petr DVOŘÁK a Albano MELI. Molecular and Functional Characterization of Uniform-Sized Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells. In 58th Annual Meeting of the Biophysical-Society. 2014. ISSN 0006-3495. Dostupné z: https://dx.doi.org/10.1016/j.bpj.2013.11.3135.

@proceedings{1215616,
   author = {Pešl, Martin and Aćimović, Ivana and Přibyl, Jan and Héžová, Renata and Vilotić, Aleksandra and Aimond, Franck and Fauconnier, Jeremy and Vrbský, Jan and Kružliak, Peter and Skládal, Petr and Kára, Tomáš and Rotrekl, Vladimír and Lacampagne, Alain and Dvořák, Petr and Meli, Albano},
   booktitle = {58th Annual Meeting of the Biophysical-Society},
   doi = {http://dx.doi.org/10.1016/j.bpj.2013.11.3135},
   keywords = {human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium},
   language = {eng},
   note = {Accession Number: WOS:000337000403197},
   title = {Molecular and Functional Characterization of Uniform-Sized Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells},
   url = {http://www.cell.com/biophysj/pdf/S0006-3495%2813%2904394-4.pdf},
   year = {2014}
}

TY  - CONF
ID  - 1215616
AU  - Pešl, Martin - Aćimović, Ivana - Přibyl, Jan - Héžová, Renata - Vilotić, Aleksandra - Aimond, Franck - Fauconnier, Jeremy - Vrbský, Jan - Kružliak, Peter - Skládal, Petr - Kára, Tomáš - Rotrekl, Vladimír - Lacampagne, Alain - Dvořák, Petr - Meli, Albano
PY  - 2014
TI  - Molecular and Functional Characterization of Uniform-Sized Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells
N1  - Accession Number: WOS:000337000403197
KW  - human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium
UR  - http://www.cell.com/biophysj/pdf/S0006-3495%2813%2904394-4.pdf
N2  - In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are able to differentiate into functional cardiomyocytes (CMs). Traditional suspension method for 3D embryoid bodies (EBs) formation results in heterogeneous EB population. Here, we hypothesized that forming homogenous EB in size and shape allows studying the mechano-biological properties of spontaneously beating clusters containing CMs. Using a defined number of single cells in AggreWell plate, we formed homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors. We checked the expression of cardiac markers by qRT-PCR, immunocytochemistry and western blotting in both beating EBs and enzymatically-dissociated CMs and found increased expression of MYH6, MYH7 and RYR2 genes as well as sarcomeric pattern for cardiac troponin T and alpha-actinin. Using atomic force microscopy-based technique, we measured the beating properties of hESC-EBs and hiPSC-EBs and found a slower beat rate in hESC-CMs when compared to hiPSC-CMs (51 ± 5 vs 74 ± 7 bpm) and similar contraction force (31 ± 7 vs 39 ± 9 nN). Both cell types respond upon stimulation or inhibition of beta-adrenergic pathway and caffeine. Using Ca2+ imaging we also demonstrated the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum and evaluated the contribution of IP3R. Thus, upon xestospongin C and histamine (IP3R modulators), spontaneous Ca2+ transients are maintained confirming the role of RyR2. Using the patch-clamp technique, we evaluated the cardiac population constituting the EBs and found that more than 75% of excitable cells exhibit atrial-like action potentials. Our results indicated that the mechano-biological properties of homogenous beating EBs can be investigated. They also indicated that spontaneous beating EBs contain mostly functional and organized atrial CMs.
ER  -

PEŠL, Martin, Ivana A$\backslash$'CIMOVI$\backslash$'C, Jan PŘIBYL, Renata HÉŽOVÁ, Aleksandra VILOTI$\backslash$'C, Franck AIMOND, Jeremy FAUCONNIER, Jan VRBSKÝ, Peter KRUŽLIAK, Petr SKLÁDAL, Tomáš KÁRA, Vladimír ROTREKL, Alain LACAMPAGNE, Petr DVOŘÁK a Albano MELI. Molecular and Functional Characterization of Uniform-Sized Beating Embryoid Bodies and Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells. In \textit{58th Annual Meeting of the Biophysical-Society}. 2014. ISSN~0006-3495. Dostupné z: https://dx.doi.org/10.1016/j.bpj.2013.11.3135.

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