Conditional immortalization of mesenchymal stem cells and their extracellular vesicles therapy for interstitial cystitis/bladder pain syndrome.

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.