Basic Study
Copyright ©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. May 26, 2024; 16(5): 551-559
Published online May 26, 2024. doi: 10.4252/wjsc.v16.i5.551
Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells
Amani Yehya, Joseph Azar, Mohamad Al-Fares, Helene Boeuf, Wassim Abou-Kheir, Dana Zeineddine, Ola Hadadeh
Amani Yehya, Joseph Azar, Mohamad Al-Fares, Wassim Abou-Kheir, Ola Hadadeh, Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Beirut 1107-2020, Lebanon
Helene Boeuf, Inserm, Biotis, U1026, University Bordeaux, Bordeaux F-33000, France
Dana Zeineddine, Rammal Rammal Lab, Physio-Toxicity Environmental Team, Faculty of Sciences, Lebanese University, Nabatieh 0000, Lebanon
Co-first authors: Amani Yehya and Joseph Azar.
Co-corresponding authors: Dana Zeineddine and Ola Hadadeh.
Author contributions: Yehya A and Azar J contributed equally to this work as co-first authors, and they wrote the manuscript and performed data analysis and interpretation; Al-Fares M performed initial lab work and participated in data collection; Boeuf H provided the genetically manipulated cell lines employed in this study; Abou-Kheir W, Zeineddine D, and Hadadeh O contributed as co-senior authors, and they conceived the project, managed and supervised the work, discussed the data, acquired the reagents, and corrected the manuscript draft; Zeineddine D and Hadadeh O contributed as co-corresponding authors, and they acquired the funding and reagents; and all authors have reviewed and approved the final manuscript.
Supported by the National Council for Scientific Research in Lebanon, CNRS-L.
Institutional review board statement: This is to confirm that no patient samples were used in this research study.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Ola Hadadeh, PhD, Associate Research Scientist, Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Bliss Street-Hamra, Beirut 1107-2020, Lebanon. hadadeh.ola@gmail.com
Received: December 30, 2023
Revised: January 29, 2024
Accepted: April 1, 2024
Published online: May 26, 2024
Processing time: 145 Days and 10.2 Hours
Abstract
BACKGROUND

Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood.

AIM

To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2, Pim-2, and Met-1, respectively.

METHODS

mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays.

RESULTS

Our findings showed that the upregulation of Bcl-2, Pim-2, and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.

CONCLUSION

Our findings revealed that the upregulation of Bcl-2, Pim-2, and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.

Keywords: Mouse embryonic stem cells; Self-renewal; Apoptosis; Cardiac differentiation; B-cell lymphoma 2; PIM-2; Metallothionein-1

Core Tip: Embryonic stem cells (ESCs) exhibit unique characteristics of self-renewal and pluripotency, allowing for their spontaneous differentiation into multiple cell lineages. Apoptotic signaling presents one of the crucial networks that influence embryonic development. In this study, the upregulation of anti-apoptotic genes B-cell lymphoma 2, Pim-2, and metallothionein-1 in mouse ESCs altered cardiac differentiation. This alteration was evidenced by a decreased size of embryoid bodies, a reduced number of beating cardiomyocytes, and an attenuated expression of cardiac markers. The study emphasizes the critical role of apoptosis in cardiac differentiation, providing insights into the regulatory impact of apoptosis on early differentiation pathways.