Abstract
Autologous endothelial progenitor cells (EPCs) might be alternative angiogenic cell sources for vascularization of tissue-engineered bladder, while isolation and culture of EPCs from peripheral blood in adult are usually time-consuming and highly inefficient. Recent evidence has shown that EPCs also exist in the adipose tissue. As adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method, the aim of this study was to culture and characterize EPCs from adipose tissue (ADEPCs) and investigate their potential for the neovascularization of tissue-engineered bladder. Adipose stromal vascular fraction (SVF) was isolated and used for the culture of ADEPCs and adipose derived stem cells (ADSCs). After SVF was cultured for one week, ADEPCs with typical cobblestone morphology emerged and could be isolated from ADSCs according to their different responses to trypsinization. Rat bladder smooth muscle cells (RBSMCs) were isolated and cultured from rat bladder. RBSMCs exhibited typical spindle-shaped morphology. ADEPCs had higher proliferative potential than ADSCs and RBSMCs. ADEPCs stained positive for CD34, Stro-1, VEGFR-2, eNOS and CD31 but negative for α-SMA, CD14 and CD45. ADSCs stained positive for CD34, Stro-1 and α-SMA but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. RBSMCs stained only positive for α-SMA. ADEPCs could be expanded from a single cell at an early passage to a cell cluster containing more than 10,000 cells. ADEPCs were able to uptake DiI-Ac-LDL, bind UEA-1 and form capillary-like structures in three-dimensional scaffolds (Matrigel and bladder acellular matrix). ADEPCs were also able to enhance the human umbilical vein endothelial cells’ capability of capillary-like tube formation on Matrigel. Additionally, significantly higher levels of mRNA and protein of vascular endothelial growth factor were found in ADEPCs than in RBSMCs. These results suggest the potential use of ADEPCs as angiogenic cell sources for engineering bladder tissue.
Highlights
Many patients suffering from congenital and acquired diseases such as exstrophy, trauma, inflammation and cancer, often end up with impairment of bladder structure and function, and eventually are in need of bladder reconstruction
When the adipose derived stem cells (ADSCs) were dissociated during trypsinization, the endothelial-like adipose tissue derived EPCs (ADEPCs) colonies still adhered to the flask surface and allowed to grow until confluence (Fig. 1C)
We found that ADEPCs but not ADSCs could incorporate DiI-Ac-LDL and bind UEA-1 (Fig. 5A)
Summary
Many patients suffering from congenital and acquired diseases such as exstrophy, trauma, inflammation and cancer, often end up with impairment of bladder structure and function, and eventually are in need of bladder reconstruction. There are still several challenges ahead of us that need to be completely resolved before this technique is widely applied in clinic[3]. Vascularization of engineered bladder tissue is one of the most urgent challenges in tissue engineering of the bladder. Prevascularization of the engineered construct in vitro using autologous endothelial cells might be a novel approach for the rapid establishment of adequate blood supply after bladder reconstruction[6]. Isolation and culture of endothelial cells usually requires an invasive procedure for vessel harvest, which may lead to donor-site morbidity. A relatively less invasive procedure for obtaining autologous cells is highly desirable [7]
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