Differentiated adipose-derived stem cells for bladder bioengineering

Abstract

Objective. The aim of this study was to characterize and differentiate adipose-derived stem cells (ADSCs) to functional smooth muscle cells (SMCs) as an alternative cell source for bladder engineering. Materials and methods. Rat ADSCs were differentiated into SMCs for 1-6 weeks using induction medium. The changes in contractile genes and protein expression were investigated by real-time polymerase chain reaction, fluorescence-activated cell sorting and Western blot at different time-points. Spontaneous and carbachol-induced contractions of engineered SMC tissue at different stages were investigated to define the optimal duration of induction. Results. ADSCs differentiated into SMCs lost their capacity for expansion and their contractile phenotype, changing to a synthetic phenotype over time. Highest levels of calponin, smoothelin and MyH11 expression were observed in ADSCs induced for 3 weeks. Cells acquired typical SMC morphology when contractile proteins were expressed. However, SMC morphology was lost with reduction of contractile proteins, especially smoothelin and MyH11. The maximal spontaneous and carbachol-induced contraction of differentiated ADSCs was after 3 weeks. Conclusions. This study demonstrates that ADCSs are a suitable cell source for engineering tissues that require functional and contractile SMCs. An induction time of 3 weeks appears to be sufficient for ADSC differentiation to contractile SMCs suitable for urological tissue engineering. Materials and MethodsRat ADSCs were differentiated into SMCs for 1 to 6 weeks using induction medium. The changes in contractile genes and proteins expression were investigated by Real time PCR, FACS, and western blot (WB) at different time points. In addition, spontaneous and carbachol-induced contractions of engineered SMC tissue at different stages were investigated to define the optimal duration of induction. ResultsWe found that ADSCs differentiated into SMCs lose their capacity for expansion and their contractile phenotype changing to a synthetic phenotype over time.Highest levels of calponin, smoothelin, and MyH11 expression were observed in ADSCs induced for 3 weeks. Cells acquired typical SMC morphology when contractile proteins were expressed. However, SMC morphology was lost with reduction of contractile proteins, especially smoothelin and MyH11. The maximal spontaneous and carbachol-induced contraction of differentiated ADSC was after 3 weeks. Conclusions 2Our study demonstrates that ADCSs are a suitable cell source for engineering tissues that require functional and contractile SMCs. An induction time of 3 weeks appears to be sufficient for ADSC differentiation to contractile SMCs suitable for urologic tissue engineering.

Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting

“…Therefore, hADSCs have become an attractive stem cell source for tissue engineering. Many researchers have investigated the smooth muscle differentiation of hADSCs due to their promising applications in the field of cellular therapies involving urinary and cardiovascular systems (Choi et al, 2010; Fu et al, 2010; Salemi et al, 2015). These studies provided various methods for smooth muscle cell differentiation, including co-culturing hADSCs with primary myoblasts using heparin, 5-azacytidine or transforming growth factor β1 (TGF-β1) as smooth muscle inducing factors and attaching hADSCs to microcarriers created by thermally induced phase separation (TIPS; Di Rocco et al, 2006; Rodriguez et al, 2006; Meligy et al, 2012; Park et al, 2013; Parmar et al, 2015).…”

Increased autophagy contributes to impaired smooth muscle function in neurogenic lower urinary tract dysfunction

“…For negative controls, the primary antibody was omitted. The specificity of the commercial antibodies has been thoroughly validated in our former studies …”

“…The specificity of the commercial antibodies has been thoroughly validated in our former studies. 19,20 2.6 | Pharmacological modulation of autophagy Biopsy-derived SMCs were cultured in the presence of 2 mM and 10 mM 3-methyladenine (3-MA) (Sigma-Aldrich, Buchs, Switzerland), a pharmacological inhibitor commonly used to block autophagy. 21 Treatment of the cells with 2 mM 3-MA was performed under culture conditions after 7 days and the treatment with 10 mM 3-MA was for 24 h only.…”

The effect of a cyclic uniaxial strain on urinary bladder cells

“…Recently, stem cells derived from urine or adipose tissue have been successfully differentiated to functional smooth muscle cells for bladder tissue engineering applications. In addition, these stem cells can be harvested from the patient relatively easy, providing an autologous cell source [ 25 , 26 ].…”