Basic Study
Copyright ©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Apr 26, 2024; 16(4): 410-433
Published online Apr 26, 2024. doi: 10.4252/wjsc.v16.i4.410
Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine
Shafiqa Naeem Rajput, Bushra Kiran Naeem, Anwar Ali, Asmat Salim, Irfan Khan
Shafiqa Naeem Rajput, Asmat Salim, Irfan Khan, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
Bushra Kiran Naeem, Surgical Unit 4, Dr. Ruth KM Pfau Civil Hospital, Karachi 74400, Pakistan
Anwar Ali, Department of Physiology, University of Karachi, Karachi 75270, Pakistan
Irfan Khan, Center for Regenerative Medicine and Stem Cells Research, and Department of Ophthalmology and Visual Sciences, The Aga Khan University, Karachi 74800, Sindh, Pakistan
Author contributions: Rajput SN performed experiments and wrote the original manuscript; Naeem BK helped in data evaluation and writing; Ali A contributed to the experimentation and writing; Salim A reviewed the manuscript; Salim A and Khan I evaluated and analyzed the data; Khan I conceived and designed the studies and finalized the manuscript.
Supported by Higher Education Commission, Islamabad, Pakistan grant, No. 20-17590/NRPU/R&D/HEC/2021 2021.
Institutional review board statement: The ethical approval for the present study was obtained from the Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi (IEC document No. 40/2020/QD-VINMEC) in accordance with the Helsinki Declaration.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: All the data is presented in the manuscript. The raw data will be provided upon request.
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: Irfan Khan, PhD, Assistant Professor, Center for Regenerative Medicine and Stem Cells Research, and Department of Ophthalmology and Visual Sciences, The Aga Khan University, Stadium Road, P.O. Box 3500, Karachi 74800, Sindh, Pakistan. irfankhan.bangash@aku.edu
Received: November 30, 2023
Peer-review started: November 30, 2023
First decision: January 15, 2024
Revised: February 1, 2024
Accepted: March 18, 2024
Article in press: March 18, 2024
Published online: April 26, 2024
Processing time: 146 Days and 16.4 Hours
ARTICLE HIGHLIGHTS
Research background

Stem cells are at the forefront for their intriguing potential in regenerative medicine. Organ or tissue regeneration requires 10 to 400 million mesenchymal stem cells (MSCs) per dose for transplantation. MSCs are undifferentiated cells with the potential to self-renew, capability to differentiate into multiple lineages, and immune modulation potential, which is considered ideal for cell-based therapeutics. The ex-vivo propagation and proliferation of MSCs are vital to fulfill the requisite cell number. Despite the current challenges in cell-based therapies, the safety levels of umbilical cord tissue derived MSCs render them a promising therapeutic approach in regenerative medicine.

Research motivation

MSC expansion is associated with extensive subculturing and cell passage. However, genetic instability during their ex-vivo expansion makes them less efficacious due to senescence. MSCs from different donors result in tissue vs graft rejection during transplantation. Recultured human umbilical cord (hUC) improves the culturing capability of MSCs for expansion and could provide novel sight for cell-based therapeutic applications which could be economical to maintain good manufacturing practices.

Research objectives

The objective of the proposed study was to establish an efficacious method for ex-vivo expansion of hUC-MSCs.

Research methods

MSCs from the hUC were isolated, and the hUC explant was recultured to get maximum number of cells. Isolated hUC-MSC at every recultured group were characterized on the bases of morphology. Immunophenotyping of positive and negative markers expressed by MSC, immunocytochemical staining and trilineage differentiation. The regulatory genes specific to the MSCs stemness, proliferation, and senescence markers were determined at every recultured group. The senescence was evaluated by beta galactosidase staining. The recultured hUC-MSCs were quantitatively characterized by population doubling time, population doubling number, colony forming unit, and cell cycle analysis. The absence of mycoplasma, cytomegalovirus, and endotoxin was evaluated in recultured hUC isolated MSC.

Research results

Recultured hUC derived MSCs interestingly showed no significant difference between each recultured hUC-MSC for pluripotent gene expression markers. These expanded MSCs showed positive immunophenotypic markers and lacked the expression of negative markers. Simultaneously, recultured hUC-MSCs showed positive expression for multilineage differentiation into osteogenic, chondrogenic, and adipogenic lineages. The proliferation of recultured hUC-MSCs was observed until passage 15. Nevertheless, a significantly increased mitotic phase of the cell cycle was observed in all recultured hUC-MSCs groups. Delayed cellular senescence was observed by increased expression of human telomerase reverse transcriptase (hTERT). The senescence-associated β-galactosidase activity was not detected in repeatedly recultured hUC-MSCs. The quality of recultured hUC-MSCs was maintained and showed negative expression of mycoplasma, cytomegalovirus, and endotoxin.

Research conclusions

This study leads to the development of a novel protocol for scaling stem cells population. In-vitro expansion of the cell dose required for in-vivo implantation could be achieved in a short time. It can decrease tissue versus graft rejection due to the less rigorous HLA screening performed in stem cell banks. Since these MSCs were isolated from the same recultured hUC, they thus exhibit enduring MSC stemness, and adhere with the recommendation of International Society of Cellular Therapy.

Research perspectives

This approach will help to isolate and propagate a larger number of cells required for clinical applications, and translational medicine. This approach will enable the faster availability of the required number of cells from the same initial source, thus reducing the need for extensive donor screening and resource allocation. This protocol offers cost effectiveness by potentially reducing reliance on multiple donors and complex HLA matching procedure.