Characterization of smooth muscle cells and urothelial cells differentiated from human adipose derived stem cells for seeding a bladder tissue biomimetic patch.

摘要

Approximately 400 million people worldwide suffer from bladder disease, which can lead to the development of high pressure and low-compliance bladders. Current surgical solutions include replacing diseased bladder tissue with a segment of gastrointestinal (GI) tissue, referred to as a bladder augmentation. However, GI segments are known to cause a myriad of issues when implanted within the bladder, including urinary tract infection, metabolic abnormalities and abnormal drug kinetics. The objective of the present study is to investigate hADSCs as a cell source to seed a biomimetic bladder tissue patch for the purpose of bladder tissue remodeling and regeneration. hADSCs were exposed to smooth muscle inductive medium (SMIM) to induce smooth muscle cell (SMC) differentiation. Differentiated SMCs and urothelial cells were also cultured in non-inductive growth media to determine the level of differentiation. Changes at the morphological, mRNA and protein level were observed through phase-contrast imaging, RT-PCR and immunofluorescence. Differentiated SMCs and urothelial cells were co-cultured in varying media conditions to determine the effect of each cell type on the retention of the other's differentiated traits, and changes at the protein level were observed. Differentiated SMCs were also cultured on fibronectin-gelatin coated PCUU scaffolds to determine the SMCs ability to infiltrate the scaffold. After culture in SMIM for 10 days, ADSCs exhibited increased mRNA expression of alphaSMA, SM-22alpha and SM-MHC and decreased expression of CD90 and CD105. Protein expression of alphaSMA, SM-22alpha and SM-MHC was also exhibited after incubation in SMIM. Culture in non-inductive media led to the decrease in mRNA expression of alpha-SMA, SM-22alpha and SM-MHC and UP1b in SMCs and urothelial cells, respectively, while CK20 remained unchanged. mRNA expression of CD90 and CD105 was increased after incubation in non-inductive media in both dedifferentiated SMCs and urothelial cells. At the protein level, expression of SM-MHC and UP1b was lost in dedifferentiated SMCs and urothelial cells, respectively, while expression of alpha-SMA, SM-22alpha and CK20 was retained. After incubation in a co-culture, protein expression of SM-MHC and UP1b was lost in SMCs and urothelial cells, respectively, while expression of alpha-SMA, SM-22alpha and CK20 was retained. Differentiated SMCs seeded onto a coated PCUU scaffold infiltrated the scaffold to a greater degree than SMCs seeded onto an uncoated scaffold. Results of the present study indicate that soluble factors present in the cellular microenvironment have a pronounced effect on differentiation. In the present study, culturing ADSCs in SMIM was proven to be an effective method in differentiating toward a SMC lineage. However, differentiated SMCs and urothelial cells dedifferentiated once factors important to differentiation were removed. Co-culturing of differentiated SMCs and urothelial cells did not support retention of differentiated traits either. Taken together, these in vitro results indicate that cells differentiated via soluble factors within the media only achieve partial differentiation. When the differentiated SMCs were seeded on a PCUU scaffold, layer-by-layer coating with fibronectin and gelatin enhanced the infiltration into the scaffold. This is likely due to the adsorption of fibronectin to the scaffold and subsequent interaction with gelatin provides cellular adhesion sites both within and on the surface of the scaffold.