Abstract
Smooth muscle differentiated human adipose derived stem cells (hADSCs) provide a crucial stem cell source for urinary tissue engineering, but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome, including a relatively long induction time and equipment dependence, which limits access to abundant stem cells within a short period of time for further application. Three-dimensional (3D) bioprinting holds great promise in regenerative medicine due to its controllable construction of a designed 3D structure. When evenly mixed with bioink, stem cells can be spatially distributed within a bioprinted 3D structure, thus avoiding drawbacks such as, stem cell detachment in a conventional cell-scaffold strategy. Notwithstanding the advantages mentioned above, cell viability is often compromised during 3D bioprinting, which is often due to pressure during the bioprinting process. The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure. Here, we employed the hanging-drop method to generate hADSC microtissues in a smooth muscle inductive medium containing human transforming growth factor β1 and bioprinted the induced microtissues onto a 3D structure. After 3 days of smooth muscle induction, the expression of α-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs, as confirmed by immunofluorescence and western blotting analysis. The semi-quantitative assay showed that the expression of α-smooth muscle actin (α-SMA) was 0.218 ± 0.077 in MTs and 0.082 ± 0.007 in Controls; smoothelin expression was 0.319 ± 0.02 in MTs and 0.178 ± 0.06 in Controls. Induced MTs maintained their phenotype after the bioprinting process. Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time points compared to the conventional single-cell bioprinting strategy (mean cell viability was 88.16 ± 3.98 vs. 61.76 ± 15% for microtissues and single-cells, respectively). These results provide a novel way to enhance the smooth muscle differentiation of hADSCs and a simple method to maintain better cell viability in 3D bioprinting.
Highlights
Human adipose derived stem cells are known for their multilineage differentiation potential, including bone, cartilage, adipose tissue, and smooth muscle (Bajek et al, 2016)
The results showed that Human adipose derived stem cells (hADSCs) presented a strong positivity for CD44 and CD105, while no signal was detected for CD45 and CD34
A large number of hADSCs could be isolated by type I collagenase digestion from adipose tissue
Summary
Human adipose derived stem cells (hADSCs) are known for their multilineage differentiation potential, including bone, cartilage, adipose tissue, and smooth muscle (Bajek et al, 2016). 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). We performed a novel inducement procedure by combining inducing factor TGFβ1 with the hanging-drop method to induce smooth muscle differentiation while generating hADSC microtissues
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