BIOMECHANICS OF STEM CELL DERIVED CARDIOMYOCYTES CAN BE
DESCRIBED BY  ATOMIC FORCE MICROSCOPY	Optimization of
cardiomyocytes differentiation from pluripotent stem cells
using atomic force microscopy

D 2014

BIOMECHANICS OF STEM CELL DERIVED CARDIOMYOCYTES CAN BE DESCRIBED BY ATOMIC FORCE MICROSCOPY Optimization of cardiomyocytes differentiation from pluripotent stem cells using atomic force microscopy

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

Základní údaje

Originální název

BIOMECHANICS OF STEM CELL DERIVED CARDIOMYOCYTES CAN BE DESCRIBED BY ATOMIC FORCE MICROSCOPY Optimization of cardiomyocytes differentiation from pluripotent stem cells using atomic force microscopy

Autoři

PEŠL, Martin, Jan PŘIBYL, Ivana AĆIMOVIĆ, Aleksandra VILOTIĆ, Renata HÉŽOVÁ, Petr SKLÁDAL, Tomáš KÁRA, Vladimír ROTREKL, Alain LACAMPAGNE, Petr DVOŘÁK a Albano MELI

Vydání

ABSTRACT BOOK “New Challenges in the World Science: Joint Approaches of Young Scientists” 2014

Nakladatel

The Ministry of Youth and Sport of the Republic of Azerbaijan

Další údaje

Jazyk

angličtina

Typ výsledku

Stať ve sborníku

Obor

Genetika a molekulární biologie

Stát vydavatele

Ázerbájdžán

Utajení

není předmětem státního či obchodního tajemství

Forma vydání

tištěná verze "print"

Odkazy

URL

Organizační jednotka

Lékařská fakulta

Klíčová slova česky

human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium

Klíčová slova anglicky

human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 11. 1. 2015 14:44, MUDr. Martin Pešl, Ph.D.

Anotace

V originále

EXTENDED ABSTRACT Stem cell derived cardiomyocytes (SCs-CMs).can be prepared by differentiation in vitro from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Routinely is checked the expression of cardiac markers by qRT-PCR, immunocytochemistry and western blotting in beating EBs and enzymatically-dissociated CMs. Electrophysiological methods serve well fro conduction and excitation studies, but cannot reflex the mechanics. The contractions of cell clusters resembles functionality, but remains to be studied in detail. In this study, we present quantification method of the spontaneous contraction of SCs-CMs. Moreover, traditional suspension method for 3D clusters - embryoid bodies (EBs) formation results in heterogeneous EB population and heterogenouse cardiac clusters after differentiation, mainly different in size. But formation of EBs through forced aggregation of the cells allows highly uniform population of clusters. This enables involvement of Atomic force microscopy (AFM) when cantilever is directly placed on the contracting cluster. It enables to reflex the laser beam according to the movement and record it on photodiode in real time, thus create equivalent of mechanocardiogram (MCG). Methods: Using a defined number of undifferentiated single cells in AggreWell plate, we formed homogeneous EBs (2000 cells per cluster). The differentiation process is achieved using defined growth factors at different stages to enhance mesodermal differentiation and production of cardiac progenitors. Molecular and functional characterization is then achieved to confirm the cardiac identity of the resulting cells (MYH6, MYH7 and RYR2 genes as well as sarcomeric pattern for cardiac troponin T and alpha-actinin). Atomic force microscopy was performed in standard (Tyrod) media and after inhibition as well as activation to of beta-adrenergic receptors. Temperature sensitivity was tested as continuous controlled cooling of cultivation media, stepwise at 0,5 degree of Celsius in range of 37oC – 27oC. Results: Contrary to suspension method, the forced aggregation using AggreWell plates allows production of highly uniform-sized EBs with up to 80% of contracting clusters. Produced hESC- CMs and hiPSC-CMs expressed cardiac specific markers. Using atomic force microscopy-based technique, we measured the beating properties of hESC-EBs and hiPSC-EBs. A slower beat rate in hESC-CMs was obtained when compared to hiPSC-CMs (51 ± 5 vs 74 ± 7 bpm), while contracting force remained similar (31 ± 7 vs 39 ± 9 nN). Both cell types respond comparably upon stimulation or inhibition of beta-adrenergic pathway and caffeine. Conclusion: Our results indicate that the mechano-biological properties of homogenous beating EBs can be investigated by atomic force microscopy and thus stem cell derived cardiomyocytes can serve as well described disease and drug testing model. The method allowed us to optimize the cultivation and differentiation method reaching highly uniform CMs with high efficiency. REFERENCE: PEŠL, Martin, Ivana AĆIMOVIĆ, Jan PŘIBYL, Renata HÉŽOVÁ, Aleksandra VILOTIĆ, Jeremy FAUCONNIER, Jan VRBSKÝ, Peter KRUZLIAK, Petr SKLÁDAL, Tomáš KÁRA, Vladimír ROTREKL, Alain LACAMPAGNE, Petr DVOŘÁK a Albano MELI. Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells. Heart Vessels, Japan: Springer Japan, 2013. ISSN 0910-8327. doi:10.1007/s00380-013-0436-9.

Návaznosti

ED1.1.00/02.0068, projekt VaV

Název: CEITEC - central european institute of technology

EE2.3.20.0011, projekt VaV

Název: Centrum výzkumu pluripotentních buněk a nestability genomu

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, Jan PŘIBYL, Ivana AĆIMOVIĆ, Aleksandra VILOTIĆ, Renata HÉŽOVÁ, Petr SKLÁDAL, Tomáš KÁRA, Vladimír ROTREKL, Alain LACAMPAGNE, Petr DVOŘÁK a Albano MELI. BIOMECHANICS OF STEM CELL DERIVED CARDIOMYOCYTES CAN BE DESCRIBED BY ATOMIC FORCE MICROSCOPY Optimization of cardiomyocytes differentiation from pluripotent stem cells using atomic force microscopy. In ABSTRACT BOOK “New Challenges in the World Science: Joint Approaches of Young Scientists”. The Ministry of Youth and Sport of the Republic of Azerbaijan, 2014.

@inproceedings{1215609,
   author = {Pešl, Martin and Přibyl, Jan and Aćimović, Ivana and Vilotić, Aleksandra and Héžová, Renata and Skládal, Petr and Kára, Tomáš and Rotrekl, Vladimír and Lacampagne, Alain and Dvořák, Petr and Meli, Albano},
   booktitle = {ABSTRACT BOOK “New Challenges in the World Science: Joint Approaches of Young Scientists”},
   keywords = {human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium},
   howpublished = {tištěná verze "print"},
   language = {eng},
   publisher = {The Ministry of Youth and Sport of the Republic of Azerbaijan},
   title = {BIOMECHANICS OF STEM CELL DERIVED CARDIOMYOCYTES CAN BE DESCRIBED BY ATOMIC FORCE MICROSCOPY Optimization of cardiomyocytes differentiation from pluripotent stem cells using atomic force microscopy},
   url = {http://bakuworldscienceforum.com/},
   year = {2014}
}

TY  - JOUR
ID  - 1215609
AU  - Pešl, Martin - Přibyl, Jan - Aćimović, Ivana - Vilotić, Aleksandra - Héžová, Renata - Skládal, Petr - Kára, Tomáš - Rotrekl, Vladimír - Lacampagne, Alain - Dvořák, Petr - Meli, Albano
PY  - 2014
TI  - BIOMECHANICS OF STEM CELL DERIVED CARDIOMYOCYTES CAN BE DESCRIBED BY ATOMIC FORCE MICROSCOPY Optimization of cardiomyocytes differentiation from pluripotent stem cells using atomic force microscopy
PB  - The Ministry of Youth and Sport of the Republic of Azerbaijan
KW  - human pluripotent stem cell embryoid body differentiation cardiomyocyte calcium
UR  - http://bakuworldscienceforum.com/
N2  - EXTENDED ABSTRACT Stem cell derived cardiomyocytes (SCs-CMs).can be prepared by differentiation in vitro from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Routinely is checked the expression of cardiac markers by qRT-PCR, immunocytochemistry and western blotting in beating EBs and enzymatically-dissociated CMs. Electrophysiological methods serve well fro conduction and excitation studies, but cannot reflex the mechanics. The contractions of cell clusters resembles functionality, but remains to be studied in detail. In this study, we present quantification method of the spontaneous contraction of SCs-CMs. Moreover, traditional suspension method for 3D clusters - embryoid bodies (EBs) formation results in heterogeneous EB population and heterogenouse cardiac clusters after differentiation, mainly different in size. But formation of EBs through forced aggregation of the cells allows highly uniform population of clusters. This enables involvement of Atomic force microscopy (AFM) when cantilever is directly placed on the contracting cluster. It enables to reflex the laser beam according to the movement and record it on photodiode in real time, thus create equivalent of mechanocardiogram (MCG). Methods: Using a defined number of undifferentiated single cells in AggreWell plate, we formed homogeneous EBs (2000 cells per cluster). The differentiation process is achieved using defined growth factors at different stages to enhance mesodermal differentiation and production of cardiac progenitors. Molecular and functional characterization is then achieved to confirm the cardiac identity of the resulting cells (MYH6, MYH7 and RYR2 genes as well as sarcomeric pattern for cardiac troponin T and alpha-actinin). Atomic force microscopy was performed in standard (Tyrod) media and after inhibition as well as activation to of beta-adrenergic receptors. Temperature sensitivity was tested as continuous controlled cooling of cultivation media, stepwise at 0,5 degree of Celsius in range of 37oC – 27oC. Results: Contrary to suspension method, the forced aggregation using AggreWell plates allows production of highly uniform-sized EBs with up to 80% of contracting clusters. Produced hESC- CMs and hiPSC-CMs expressed cardiac specific markers. Using atomic force microscopy-based technique, we measured the beating properties of hESC-EBs and hiPSC-EBs. A slower beat rate in hESC-CMs was obtained when compared to hiPSC-CMs (51 ± 5 vs 74 ± 7 bpm), while contracting force remained similar (31 ± 7 vs 39 ± 9 nN). Both cell types respond comparably upon stimulation or inhibition of beta-adrenergic pathway and caffeine. Conclusion: Our results indicate that the mechano-biological properties of homogenous beating EBs can be investigated by atomic force microscopy and thus stem cell derived cardiomyocytes can serve as well described disease and drug testing model. The method allowed us to optimize the cultivation and differentiation method reaching highly uniform CMs with high efficiency. REFERENCE: PEŠL, Martin, Ivana AĆIMOVIĆ, Jan PŘIBYL, Renata HÉŽOVÁ, Aleksandra VILOTIĆ, Jeremy FAUCONNIER, Jan VRBSKÝ, Peter KRUZLIAK, Petr SKLÁDAL, Tomáš KÁRA, Vladimír ROTREKL, Alain LACAMPAGNE, Petr DVOŘÁK a Albano MELI. Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells. Heart Vessels, Japan: Springer Japan, 2013. ISSN 0910-8327. doi:10.1007/s00380-013-0436-9.
ER  -

PEŠL, Martin, Jan PŘIBYL, Ivana A$\backslash$'CIMOVI$\backslash$'C, Aleksandra VILOTI$\backslash$'C, Renata HÉŽOVÁ, Petr SKLÁDAL, Tomáš KÁRA, Vladimír ROTREKL, Alain LACAMPAGNE, Petr DVOŘÁK a Albano MELI. BIOMECHANICS OF STEM CELL DERIVED CARDIOMYOCYTES CAN BE DESCRIBED BY ATOMIC FORCE MICROSCOPY Optimization of cardiomyocytes differentiation from pluripotent stem cells using atomic force microscopy. In \textit{ABSTRACT BOOK “New Challenges in the World Science: Joint Approaches of Young Scientists”}. The Ministry of Youth and Sport of the Republic of Azerbaijan, 2014.

Podrobný výpis o publikaci