Characterization of smooth muscle differentiation of purified human skeletal muscle-derived cells.
ABSTRACT The purpose of this study is to characterize the smooth muscle differentiation of purified human muscle-derived cells (hMDCs). The isolation and purification of hMDCs were conducted by modified preplate technique and Dynal CD34 cell selection. Smooth muscle cell differentiation was induced by the use of smooth muscle induction medium (SMIM) and low-serum medium. The gene expressions at the mRNA and protein levels of undifferentiated and differentiated hMDCs were tested by RT-PCR, Western blot and immunofluorescence studies. Western blot and immunofluorescence studies demonstrated the purified hMDCs cultured in SMIM for 4 weeks and expressed significant amount of smooth muscle myosin heavy chain (MHC) and α-smooth muscle actin (ASMA). The cells cultured in low-serum medium for 4 weeks also expressed ASMA, while the control group did not. RT-PCR analysis showed increased gene expression of smooth muscle markers, such as ASMA, Calponin, SM22, Caldesmon, Smoothelin and MHC when purified hMDCs were exposed to SMIM for 2 and 4 weeks when compared to the controls. In conclusion, we confirmed the smooth muscle differentiation capability of purified hMDCs. The gene expression of smooth muscle differentiation of purified hMDCs was characterized. These cells may be potential biomaterials for human tissue regeneration.
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ABSTRACT: Heart valve disease is a serious and growing public health problem for which prosthetic replacement is most commonly indicated. Current prosthetic devices are inadequate for younger adults and growing children. Tissue engineered living aortic valve conduits have potential for remodeling, regeneration, and growth, but fabricating natural anatomical complexity with cellular heterogeneity remain challenging. In the current study, we implement 3D bioprinting to fabricate living alginate/gelatin hydrogel valve conduits with anatomical architecture and direct incorporation of dual cell types in a regionally constrained manner. Encapsulated aortic root sinus smooth muscle cells (SMC) and aortic valve leaflet interstitial cells (VIC) were viable within alginate/gelatin hydrogel discs over 7 days in culture. Acellular 3D printed hydrogels exhibited reduced modulus, ultimate strength, and peak strain reducing slightly over 7-day culture, while the tensile biomechanics of cell-laden hydrogels were maintained. Aortic valve conduits were successfully bioprinted with direct encapsulation of SMC in the valve root and VIC in the leaflets. Both cell types were viable (81.4 ± 3.4% for SMC and 83.2 ± 4.0% for VIC) within 3D printed tissues. Encapsulated SMC expressed elevated alpha-smooth muscle actin, while VIC expressed elevated vimentin. These results demonstrate that anatomically complex, heterogeneously encapsulated aortic valve hydrogel conduits can be fabricated with 3D bioprinting. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.Journal of Biomedical Materials Research Part A 09/2012; · 2.83 Impact Factor
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ABSTRACT: We assessed the ability of muscle-derived stem cells (MDSC) to differentiate into smooth muscle cells (SMC) and their potential to promote the regeneration of smooth muscle with a vessel extracellular matrix (VECM) for tissue engineering of the ureter. MDSC were isolated, proliferated, and identified by flow cytometry. SMC phenotype differentiation was induced with a smooth muscle induction medium. Gene expression was evaluated by real-time quantitative polymerase chain reaction (PCR) and Western blot studies. The VECM was obtained by a decellularization process, and cytotoxic effects were evaluated by exposing the induced cells to a VECM extract. The induced cells were seeded onto VECM in vitro for 1 week, and then the compound grafts were used for ureter reconstitution in vivo. The grafts were obtained for histological studies at 2, 4, 8, and 16 weeks post-operation. Intravenous urography was used to evaluate renal function and ureteral patency. Flow cytometry demonstrated that the MDSC expressed Sca-1 and desmin, but did not express CD45. After induction, SMC phenotype gene expression was confirmed in the induced cells by real-time quantitative PCR and Western blot studies. VECM exhibited a nontoxic effect on the induced cells in vitro. At 16 weeks post-operation, a histological evaluation showed that multilayered urothelium and organized muscle fiber bundles had formed in the grafts. Intravenous urography demonstrated no evidence of ureteral stricture or hydroureteronephrosis. These results demonstrate that MDSC can be induced into SMC and that this was useful for promoting regeneration of smooth muscles for ureter tissue engineering.Biotechnology and Bioprocess Engineering 17(3). · 1.28 Impact Factor
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ABSTRACT: Muscle replacement for patients suffering from extensive tissue loss or dysfunction is a major objective of regenerative medicine. To achieve functional status, bioengineered muscle replacement constructs require innervation. Here we describe a method to bioengineer functionally innervated gut smooth muscle constructs using neuronal progenitor cells and smooth muscle cells isolated from adult human donors. These constructs expressed markers for contractile smooth muscle, glial cells and mature neuronal populations. The constructs responded to physiologically relevant neurotransmitters, and neural network integration was demonstrated by response to electrical field stimulation. The ability of enteric neuroprogenitor cells to differentiate into neuronal populations provides enormous potential for functional innervation of a variety of bioengineered muscle constructs. Functionally innervated muscle constructs offer a regenerative medicine based therapeutic approach to neurodegenerative disorders.Tissue Engineering Part A 12/2013; · 4.64 Impact Factor