Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.
BACKGROUND
Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes.


METHODS
We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4+ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis.


RESULTS
After 8 days in culture, 94% ± 6% of the NKX2-5GFP+cells were beating when hESCs embryonic bodies were plated on E4+ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP+cardiomyocytes were close to the E4+ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges.


CONCLUSIONS
The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony.
Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Doha, Qatar; Department of Genetic Medicine, Weill Cornell Medicine, New York, New York, USA.
Jennifer
J
Pasquier
Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Doha, Qatar.
Renuka
R
Gupta
UMR_I 02 INERIS-URCA-ULH Unité Stress Environnementaux et BIOsurveillance des milieux aquatiques, Université de Reims, Reims, France.
Damien
D
Rioult
Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Doha, Qatar; Cardiovascular Epigenetics Laboratory, Department of Genetic Medicine, Doha, Qatar.
Jessica
J
Hoarau-Véchot
Department of Physiology and Biophysics, Weill Cornell Medicine-Qatar, Doha, Qatar.
Raphael
R
Courjaret
Department of Physiology and Biophysics, Weill Cornell Medicine-Qatar, Doha, Qatar.
Khaled
K
Machaca
Heart Hospital, Hamad Medical Corporation, Doha, Qatar.
Jassim
J
Al Suwaidi
Department of Medicine, Weill Cornell Medicine, New York, New York; Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Edouard G
EG
Stanley
Department of Medicine, Weill Cornell Medicine, New York, New York.
Shahin
S
Rafii
Department of Medicine, Weill Cornell Medicine, New York, New York; Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
David A
DA
Elliott
Department of Genetic Medicine, Weill Cornell Medicine, New York, New York, USA; Cardiovascular Epigenetics Laboratory, Department of Genetic Medicine, Doha, Qatar; Heart Hospital, Hamad Medical Corporation, Doha, Qatar; Department of Medicine, Weill Cornell Medicine, New York, New York, USA. Electronic address: cha2022@med.cornell.edu.
Charbel
C
Abi Khalil
Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Doha, Qatar; Department of Genetic Medicine, Weill Cornell Medicine, New York, New York, USA. Electronic address: jat2021@med.cornell.edu.
Arash
A
Rafii
D000818:Animals; D002318:Cardiovascular Diseases / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry; D000066449:Human Embryonic Stem Cells / Q000637:transplantation*; D006801:Humans; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D013997:Time Factors
cardiomyocytes; cardiovascular disease; embryonic stem cells; endothelial cells; vascular niche