Primed Mononuclear Cell-mediated Differentiation of Stem Cells towards Urothelial and Smooth Muscle Lineages for Urethral Regeneration

Abstract

Current limitations in treating urethral dysfunction drive the need for alternative regenerative strategies to improve clinical outcomes. While advancements in 3D bioprinting enable future development of patient-specific urethral constructs, cell retrieval remains an issue as it is commonly harvested from invasive bladder biopsies. This study investigates a stem cell differentiation approach which would eliminate the need of cell retrieval from bladder biopsies and have the potential to shorten the production time of a urethral construct. The differentiation approach is based on in vitro co-culturing of stromal vascular fraction (SVF) cells with primed mononuclear cells (MNCs). The primed MNCs were activated in vitro using decellularized tissue from either urethra or corpus spongiosum to obtain a T cell population to direct cell differentiation of the adipose derived stem cells in the SVF toward urothelial cells (UCs) and smooth muscle cells (SMCs). The cells were subsequently used for 3D bioprinting of a urethral cross section, consisting of an inner ring of urethral-like cells and an outer ring of smooth muscle-like cells. Cell differentiation was assessed through morphology evaluation, protein expression and gene expression analysis. Cell morphology was evaluated by bright field microscopy, protein expression was analyzed using immunocytochemistry (ICC) and gene expression with reverse quantitative polymer chain reaction (RT-qPCR). Cell viability within the 3D bioprinted constructs were evaluated using live/dead staining. While the differentiation toward UCs showed morphological tendencies toward urothelial-like cells, UC differentiation could not be confirmed by the gene or protein expression analysis. For differentiation toward SMCs, both morphological and gene expression analysis of desmin suggested differentiation to smooth muscle-like cells, as expression was comparable to the positive control, though protein expression analysis did not confirm this. Additionally, the control containing SVF cells cultured with decellularized corpus spongiosum showed similar results, suggesting that MNCs may not be essential, though the co-culture differentiation approach demonstrated greater differentiation potential. Furthermore, live/dead staining of the 3D bioprinted constructs suggested viable cells seven days post-printing. The combined results suggest that further studies are needed to optimize and better evaluate the co-culture differentiation approach and its application in the field of urethral regeneration.