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-Véchot, Raphael Courjaret, Khaled Machaca, Jassim Al Suwaidi, Edouard G. Stanley, Shahin Rafii, David A. Elliott, Charbel Abi Khalil, Arash Rafii

Research output: Contribution to journalArticleResearchpeer-review

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
Pages (from-to)684-693
Number of pages10
JournalJournal of Heart and Lung Transplantation
Volume36
Issue number6
DOIs
Publication statusPublished - Jun 2017
Externally publishedYes

Keywords

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

Cite this

Pasquier, Jennifer ; Gupta, Renuka ; Rioult, Damien ; Hoarau-Véchot, Jessica ; Courjaret, Raphael ; Machaca, Khaled ; Al Suwaidi, Jassim ; Stanley, Edouard G. ; Rafii, Shahin ; Elliott, David A. ; Abi Khalil, Charbel ; Rafii, Arash. / Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes. In: Journal of Heart and Lung Transplantation. 2017 ; Vol. 36, No. 6. pp. 684-693.
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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-V{\'e}chot and Raphael Courjaret and Khaled Machaca and {Al Suwaidi}, Jassim and Stanley, {Edouard G.} and Shahin Rafii and Elliott, {David A.} and {Abi Khalil}, Charbel and Arash Rafii",
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language = "English",
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Pasquier, J, Gupta, R, Rioult, D, Hoarau-Véchot, J, Courjaret, R, Machaca, K, Al Suwaidi, J, Stanley, EG, Rafii, S, Elliott, DA, Abi Khalil, C & Rafii, A 2017, 'Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes' Journal of Heart and Lung Transplantation, vol. 36, no. 6, pp. 684-693. https://doi.org/10.1016/j.healun.2017.01.001

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes. / Pasquier, Jennifer; Gupta, Renuka; Rioult, Damien; Hoarau-Véchot, Jessica; Courjaret, Raphael; Machaca, Khaled; Al Suwaidi, Jassim; Stanley, Edouard G.; Rafii, Shahin; Elliott, David A.; Abi Khalil, Charbel; Rafii, Arash.

In: Journal of Heart and Lung Transplantation, Vol. 36, No. 6, 06.2017, p. 684-693.

Research output: Contribution to journalArticleResearchpeer-review

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-Véchot, Jessica

AU - Courjaret, Raphael

AU - Machaca, Khaled

AU - Al Suwaidi, Jassim

AU - Stanley, Edouard G.

AU - Rafii, Shahin

AU - Elliott, David A.

AU - Abi Khalil, Charbel

AU - Rafii, Arash

PY - 2017/6

Y1 - 2017/6

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

VL - 36

SP - 684

EP - 693

JO - Journal of Heart and Lung Transplantation

JF - Journal of Heart and Lung Transplantation

SN - 1053-2498

IS - 6

ER -