Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes

Jennifer Pasquier, Renuka Gupta, Damien Rioult, Jessica Hoarau, Raphael Jean Courjaret, Khaled Machaca, Jassim Al Suwaidi, Edouard G. Stanley, Shahin Rafii, David A. Elliott, Charbel Abi Khalil, Arash Rafii Tabrizi

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10 Citations (Scopus)

Abstract

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.

Original languageEnglish
JournalJournal of Heart and Lung Transplantation
DOIs
Publication statusAccepted/In press - 2017

Fingerprint

Cardiac Myocytes
Endothelial Cells
Coculture Techniques
Green Fluorescent Proteins
In Vitro Techniques
Human Embryonic Stem Cells
Cell Line
Computer-Assisted Image Processing
Conditioned Culture Medium
Heart Rate
Calcium

Keywords

  • Cardiomyocytes
  • Cardiovascular disease
  • Embryonic stem cells
  • Endothelial cells
  • Vascular niche

ASJC Scopus subject areas

  • Surgery
  • Pulmonary and Respiratory Medicine
  • Cardiology and Cardiovascular Medicine
  • Transplantation

Cite this

@article{166165f751f14ec39e36c134febe722b,
title = "Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes",
abstract = "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.",
keywords = "Cardiomyocytes, Cardiovascular disease, Embryonic stem cells, Endothelial cells, Vascular niche",
author = "Jennifer Pasquier and Renuka Gupta and Damien Rioult and Jessica Hoarau and Courjaret, {Raphael Jean} and Khaled Machaca and {Al Suwaidi}, Jassim and Stanley, {Edouard G.} and Shahin Rafii and Elliott, {David A.} and {Abi Khalil}, Charbel and Tabrizi, {Arash Rafii}",
year = "2017",
doi = "10.1016/j.healun.2017.01.001",
language = "English",
journal = "Journal of Heart and Lung Transplantation",
issn = "1053-2498",
publisher = "Elsevier USA",

}

TY - JOUR

T1 - Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes

AU - Pasquier, Jennifer

AU - Gupta, Renuka

AU - Rioult, Damien

AU - Hoarau, Jessica

AU - Courjaret, Raphael Jean

AU - Machaca, Khaled

AU - Al Suwaidi, Jassim

AU - Stanley, Edouard G.

AU - Rafii, Shahin

AU - Elliott, David A.

AU - Abi Khalil, Charbel

AU - Tabrizi, Arash Rafii

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

KW - Cardiomyocytes

KW - Cardiovascular disease

KW - Embryonic stem cells

KW - Endothelial cells

KW - Vascular niche

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U2 - 10.1016/j.healun.2017.01.001

DO - 10.1016/j.healun.2017.01.001

M3 - Article

JO - Journal of Heart and Lung Transplantation

JF - Journal of Heart and Lung Transplantation

SN - 1053-2498

ER -