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Updated: Sep 10, 2025
Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells
Published on: September 23, 2022
Conditional immortalization of mesenchymal stem cells and their extracellular vesicles therapy for interstitial
Guolong Liao1, Canling Long2, Rui Guo2
1Department of Urology, Kidney and Urology Center, Pelvic Floor Disorders Center, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, 518107, China.
Background:
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition characterized by debilitating pelvic pain. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are recognized as pivotal mediators of MSCs' paracrine activity and represent a novel therapeutic approach for IC/BPS. However, their efficacy is hindered by the inherent variability of primary MSCs (pMSCs) from different donors and their susceptibility to senescence during culture expansion.
Methods:
To overcome these challenges, we developed conditionally immortalized mesenchymal stem cells (iMSCs) using a doxycycline-regulated simian virus 40 Large T expression system in pMSCs. The conditioned proliferation capacity of iMSCs was evaluated using cell counting and immunocytochemistry. The expression of surface markers on iMSCs was assessed by flow cytometry. The osteogenic and adipogenic differentiation capabilities of iMSCs at different population doubling (PD) numbers were analyzed by qPCR, alizarin red staining, and oil red O staining. The EVs secreted by iMSCs were characterized using Western blot, scanning electron microscopy, and particle size analysis. In vitro and in vivo bladder inflammation models were used to evaluate the therapeutic effects of EVs on IC/BPS.
Results:
These iMSCs exhibited precisely controlled proliferation, maintained surface marker expression and differentiation capacities, comparable to pMSCs up to PD 40. The characteristics of iMSC-EVs are equivalent to those of pMSCs. Furthermore, in vitro cellular experiments demonstrate that iMSC-EVs provide protective effects against LPS/ATP-induced damage in SV-HUC-1 cells. Additionally, administration of iMSC-EVs significantly enhanced tissue healing and anti-inflammatory capabilities in an IC/BPS animal model.
Conclusions:
In summary, this approach produced a reliable source of functional MSCs and EVs, with iMSC-EVs demonstrating robust immunomodulatory properties and promoting tissue healing in IC/BPS. This method represents a promising alternative to pMSCs for IC/BPS therapy.
Insights
Conditionally immortalized mesenchymal stem cells (iMSCs) and their derived extracellular vesicles (iMSC-EVs) offer a reliable therapy for interstitial cystitis/bladder pain syndrome (IC/BPS), showing improved tissue healing and anti-inflammatory effects.
Area of Science:
- Regenerative Medicine
- Cell Therapy
- Biotechnology
Background:
- Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition causing debilitating pelvic pain.
- Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) show therapeutic potential for IC/BPS.
- Primary MSC variability and senescence limit the efficacy of current MSC-EV therapies.
Purpose of the Study:
- To develop a reliable source of functional mesenchymal stem cells (MSCs) and their derived extracellular vesicles (EVs) for IC/BPS therapy.
- To overcome the limitations of primary MSCs (pMSCs) by creating conditionally immortalized MSCs (iMSCs).
Main Methods:
- Developed iMSCs using a doxycycline-regulated SV40 Large T expression system in pMSCs.
- Evaluated iMSC proliferation, surface marker expression, and differentiation capacity.
- Characterized iMSC-EVs and assessed their therapeutic effects in vitro and in vivo IC/BPS models.
Main Results:
- iMSCs demonstrated controlled proliferation and maintained differentiation capabilities comparable to pMSCs.
- iMSC-EVs exhibited characteristics equivalent to pMSCs and provided protection against cellular damage.
- iMSC-EV administration significantly improved tissue healing and anti-inflammatory responses in an IC/BPS animal model.
Conclusions:
- This approach yields a reliable source of functional iMSCs and iMSC-EVs.
- iMSC-EVs possess robust immunomodulatory properties and promote tissue healing in IC/BPS.
- iMSC-EVs represent a promising alternative therapeutic strategy for IC/BPS.
