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

Sammanfattning

Current limitations in treating urethral dysfunction drive the need for alternativeregenerative strategies to improve clinical outcomes. While advancements in 3Dbioprinting enable future development of patient-specific urethral constructs, cell retrievalremains an issue as it is commonly harvested from invasive bladder biopsies.This study investigates a stem cell differentiation approach which would eliminatethe need of cell retrieval from bladder biopsies and have the potential to shorten theproduction time of a urethral construct. The differentiation approach is based on invitro co-culturing of stromal vascular fraction (SVF) cells with primed mononuclearcells (MNCs). The primed MNCs were activated in vitro using decellularized tissuefrom either urethra or corpus spongiosum to obtain a T cell population to directcell differentiation of the adipose derived stem cells in the SVF toward urothelialcells (UCs) and smooth muscle cells (SMCs). The cells were subsequently used for3D bioprinting of a urethral cross section, consisting of an inner ring of urethral-likecells and an outer ring of smooth muscle-like cells. Cell differentiation was assessedthrough morphology evaluation, protein expression and gene expression analysis.Cell morphology was evaluated by bright field microscopy, protein expression wasanalyzed using immunocytochemistry (ICC) and gene expression with reverse quantitativepolymer chain reaction (RT-qPCR). Cell viability within the 3D bioprintedconstructs were evaluated using live/dead staining. While the differentiation towardUCs showed morphological tendencies toward urothelial-like cells, UC differentiationcould not be confirmed by the gene or protein expression analysis. For differentiationtoward SMCs, both morphological and gene expression analysis of desmin suggesteddifferentiation to smooth muscle-like cells, as expression was comparable to the positivecontrol, though protein expression analysis did not confirm this. Additionally,the control containing SVF cells cultured with decellularized corpus spongiosumshowed similar results, suggesting that MNCs may not be essential, though theco-culture differentiation approach demonstrated greater differentiation potential.Furthermore, live/dead staining of the 3D bioprinted constructs suggested viablecells seven days post-printing. The combined results suggest that further studiesare needed to optimize and better evaluate the co-culture differentiation approachand its application in the field of urethral regeneration.