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©The Author(s) 2025. Published by Baishideng Publishing Group Inc. All rights reserved.
Impact of differentiation protocols on the functionality of mesenchymal stem cells derived from induced pluripotent stem cells
Nidaa A Ababneh, Enas Alwohoush, Razan AlDiqs, Mohammad A Ismail, Ban Al-Kurdi, Raghda Barham, Renata Al-Atoom, Fairouz Nairat, Sabal Al Hadidi, Suha Whaibi, Mohammad H Gharandouq, Suzan Zalloum, Sofian Al Shboul, Talal Al-Qaisi, Areej Abuhammad, Tareq Saleh, Abdalla Awidi
Nidaa A Ababneh, Enas Alwohoush, Razan AlDiqs, Ban Al-Kurdi, Raghda Barham, Renata Al-Atoom, Fairouz Nairat, Sabal Al Hadidi, Suha Whaibi, Mohammad H Gharandouq, Suzan Zalloum, Abdalla Awidi, Cell Therapy Center, University of Jordan, Amman 11942, Jordan
Razan AlDiqs, Department of Allied Sciences, Faculty of Arts and Sciences, Al-Ahliyya Amman University, Amman 19111, Jordan
Mohammad A Ismail, South Australian ImmunoGENomics Cancer Institute, Adelaide Medical School, University of Adelaide, Adelaide 5005, Australia
Sofian Al Shboul, Tareq Saleh, Department of Pharmacology and Public Health, Faculty of Medicine, Hashemite University, Zarqa 13133, Jordan
Talal Al-Qaisi, Department of Biomedical Sciences, College of Health Sciences, Abu Dhabi University, Abu Dhabi 59911, United Arab Emirates
Areej Abuhammad, Department of Pharmaceutical Sciences, School of Pharmacy, University of Jordan, Amman 11942, Jordan
Tareq Saleh, Department of Pharmacology & Therapeutics, College of Medicine & Health Sciences, Arabian Gulf University, Manama 329, Bahrain
Abdalla Awidi, Department of Hematology and Oncology, Jordan University Hospital, Amman 11942, Jordan
Author contributions: Ababneh NA and Awidi A conceptualized the study and overall supervision; Ababneh NA, Alwohoush E, Ismail MA, Al-Kurdi B, Barham R, Al-Atoom R, Nairat F, Al Hadidi S, Whaibi S, Gharandouq MH, and Zalloum S carried out the experiments; Alwohoush E and AlDiqs R wrote the initial draft of the manuscript; AlDiqs R, Al Shboul S, Al-Qaisi T, Abuhammad A, and Saleh T revised and edited the manuscript; Ababneh NA, AlDiqs R, and Saleh T contributed to the formal analysis, results interpretation and approved the final version of the manuscript; Abuhammad A critically revised the entire manuscript for accuracy, clarity, and integration of reviewer feedback in line with journal standards. All authors have read and approved the final version of publication.
Institutional review board statement: This study fully adheres to ethical standards and guidelines. It received approval from the Institutional Review Board (No. IRB-CTC/2-2021/09) at the Cell Therapy Center/University of Jordan and written informed consent, in accordance with the Declaration of Helsinki.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: All data are available from the corresponding author upon reasonable request. Raw flow cytometry files and detailed protocols can be shared under an institutional material transfer agreement.
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: Nidaa A Ababneh, PhD, Associate Professor, Cell Therapy Center, University of Jordan, Queen Rania Street, Amman 11942, Jordan.
n.ababneh@ju.edu.jo
Received: June 10, 2025
Revised: August 12, 2025
Accepted: October 24, 2025
Published online: December 26, 2025
Processing time: 199 Days and 4.8 Hours
BACKGROUND
The discovery of induced pluripotent stem cells revolutionized regenerative medicine, providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs).
AIM
To evaluate and compare five iMSC differentiation protocols, assessing their efficiency, phenotypic characteristics, and functional properties relative to primary mesenchymal stem cells (MSCs).
METHODS
Five iMSC differentiation protocols were assessed: SB431542-based differentiation (iMSC1, iMSC3), an iMatrix-free method (iMSC2), growth factor supplementation (iMSC4), and embryoid body formation with retinoic acid (EB-iMSC). iMSC identity was confirmed according to the International Society for Cell & Gene Therapy 2006 criteria, requiring expression of surface markers (CD105, CD73, CD90) and absence of pluripotency markers. Functional assays were conducted to evaluate differentiation potential (osteogenic and adipogenic), proliferation, mitochondrial function, reactive oxygen species, senescence, and migration.
RESULTS
All iMSC types expressed MSC markers and lacked pluripotency markers. EB-iMSC and iMSC2 showed enhanced osteogenesis (runt-related transcription factor 2; P ≤ 0.01 and P ≤ 0.0001, respectively), while adipogenic potential was reduced in iMSC2 (Adipsin; P ≤ 0.01) and EB-iMSC (Adipsin and peroxisome proliferator-activated receptor gamma; P ≤ 0.0001 and P ≤ 0.01, respectively). Proliferation was comparable or superior to bone marrow MSCs, except in iMSC1, with iMSC4 showing the highest rate (MTT assay; P values ranged from 0.01 to 0.001). Despite reduced mitochondrial health in iMSC3 and iMSC4 (P ≤ 0.001), reactive oxygen species levels were lower in all iMSCs (P values ranged from 0.001 to 0.0001), and senescence was significantly reduced in all iMSCs with the exception of iMSC1 (P values ranged from 0.01 to 0.0001). Migration was most reduced in iMSC4 (P ≤ 0.001 at 24 hours and P ≤ 0.0001 at 48 hours).
CONCLUSION
While all protocols generated functional iMSCs, variations in differentiation, proliferation, and function emphasize the impact of protocol selection. These findings contribute to optimizing iMSC generation for research and clinical applications.
Core Tip: Induced pluripotent stem cell-derived mesenchymal stem cells (MSCs) meet standard MSC criteria, yet their differentiation, proliferation, and functional properties vary considerably depending on the generation protocol. We compared five differentiation strategies, identifying distinct strengths and limitations in osteogenesis, adipogenesis, mitochondrial function, oxidative stress, senescence, and migration. Our findings highlight the need to optimize and standardize differentiation methods to produce reproducible, high-quality induced pluripotent stem cell-derived MSCs for research and therapeutic applications.
