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Cell alignment induced by anisotropic electrospun fibrous scaffolds alone has limited effect on cardiomyocyte maturation.

ABSTRACT: Enhancing the maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) will facilitate their applications in disease modeling and drug discovery. Previous studies suggest that cell alignment could enhance hPSC-CM maturation; however, the robustness of this approach has not been well investigated. To this end, we examined if the anisotropic orientation of hPSC-CMs imposed by the underlying aligned fibers within a 3D microenvironment could improve the maturation of hPSC-CMs. Enriched hPSC-CMs were cultured for two weeks on Matrigel-coated anisotropic (aligned) and isotropic (random) polycaprolactone (PCL) fibrous scaffolds, as well as tissue culture polystyrenes (TCPs) as a control. As expected, hPSC-CMs grown on the two types of fibrous scaffolds exhibited anisotropic and isotropic orientations, respectively. Similar to cells on TCPs, hPSC-CMs cultured on these scaffolds expressed CM-associated proteins and were pharmacologically responsive to adrenergic receptor agonists, a muscarinic agonist, and a gap junction uncoupler in a dose-dependent manner. Although hPSC-CMs grown on anisotropic fibrous scaffolds displayed the highest expression of genes encoding a number of sarcomere proteins, calcium handling proteins and ion channels, their calcium transient kinetics were slower than cells grown on TCPs. These results suggest that electrospun anisotropic fibrous scaffolds, as a single method, have limited effect on improving the maturation of hPSC-CMs.

SUBMITTER: Han J

PROVIDER: S-EPMC4903921 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Cell alignment induced by anisotropic electrospun fibrous scaffolds alone has limited effect on cardiomyocyte maturation.

Han Jingjia J Wu Qingling Q Xia Younan Y Wagner Mary B MB Xu Chunhui C

Stem cell research 20160418 3

Enhancing the maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) will facilitate their applications in disease modeling and drug discovery. Previous studies suggest that cell alignment could enhance hPSC-CM maturation; however, the robustness of this approach has not been well investigated. To this end, we examined if the anisotropic orientation of hPSC-CMs imposed by the underlying aligned fibers within a 3D microenvironment could improve the maturation of hPSC-CMs. Enr ...[more]

PMID: 27131761

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Project description:BackgroundTissue engineering of the annulus fibrosus (AF) shows promise as a treatment for patients with degenerative disc disease (DDD). However, it remains challenging due to the intrinsic heterogeneity of AF tissue. Fabrication of scaffolds recapitulating the specific cellular, componential, and microstructural features of AF, therefore, is critical to successful AF tissue regeneration.MethodsPoly-L-lactic acid (PLLA) fibrous scaffolds with various fiber diameters and orientation were prepared to mimic the microstructural characteristics of AF tissue using electrospinning technique. AF-derived stem cells (AFSCs) were cultured on the PLLA fibrous scaffolds for 7 days.ResultsThe morphology of AFSCs significantly varied when cultured on the scaffolds with various fiber diameters and orientation. AFSCs were nearly round on scaffolds with small fibers. However, they became spindle-shaped on scaffolds with large fibers. Meanwhile, upregulated expression of collagen-I gene happened in cells cultured on scaffolds with large fibers, while enhanced expression of collagen-II and aggrecan genes was seen on scaffolds with small fibers. The production of related proteins also showed similar trends. Further, culturing AFSCs on a heterogeneous scaffold by overlaying membranes with different fiber sizes led to the formation of a hierarchical structure approximating native AF tissue.ConclusionFindings from this study demonstrate that fibrous scaffolds with different fiber sizes effectively promoted the differentiation of AFSCs into specific cells similar to the types of cells at various AF zones. It also provides a valuable reference for regulation of cell differentiation and fabrication of engineered tissues with complex hierarchical structures using the physical cues of scaffolds.The translational potential of this articleEffective AF repair is an essential need for treating degenerative disc disease. Tissue engineering is a promising approach to achieving tissue regeneration and restoring normal functions of tissues. By mimicking the key structural features of native AF tissue, including fiber size and alignment, this study deciphered the effect of scaffold materials on the cell differentiation and extracellular matrix deposition, which provides a solid basis for designing new strategies toward more effective AF repair and regeneration.

Project description:Despite efforts to mature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) for disease modeling and high throughput screening, cells remain immature and may not reflect adult biology. Recent advancements utilize electro-mechanical and paracrine stimulation to functionally mature cardiomyocytes, but the resulting engineered constructs continue to lack a microenvironment conducive to electrical signal propagation and maturity. Conductive polymers are attractive candidates to facilitate electrical communication between gaps in sparse hPSC-CM clusters or between hPSC-CMs to repair conduction defects. To create a conductive polymer platform for improved electrical signal propagation between hPSC-CMs and achieve electrical maturity, we electrospun poly(3,4-ethylendioxythiophene):polystyrene sulfonate (PEDOT:PSS) blended with 8% (w/v) poly(vinyl alcohol) (PVA). Matrix fiber structure remained stable over 4 weeks in buffer, stiffness remains near cardiac stiffness in vivo, and electrical conductivity scaled with PEDOT:PSS concentration. When fibroblasts were added to fibers, cells had higher initial attachment to PEDOT:PSS compared to PVA-only scaffolds, and after 5 days, over 90% of fibroblasts remained viable on PEDOT:PSS scaffolds compared to PVA-only scaffolds. Electrically excitable hPSC-CMs cultured on conductive substrates exhibited an upregulation of cardiac and muscle-related genes as opposed to non-conductive substrates. These cells further displayed increased desmoplakin (DP) localization on conductive scaffolds, indicating an improvement in the mechanical stability of our hPSC-CMs. Sarcomere organization also scaled with increasing PEDOT:PSS concentration, even in sub-monolayer cell densities, suggesting that improved organization of the contractile machinery in these cells was due to the electrical condition of the matrix. Calcium handling indicated higher calcium flux with a shorter time to peak, further suggesting improved electrical maturity, even when sub-confluent. Taken together, these data suggest that PEDOT:PSS/PVA scaffolds are stable, of a stiffness relevant to cardiomyocytes, and supportive of electrical coupling even in the absence of a monolayer, which may improve cardiac disease modeling and drug development.

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Cell alignment induced by anisotropic electrospun fibrous scaffolds alone has limited effect on cardiomyocyte maturation.

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Cell alignment induced by anisotropic electrospun fibrous scaffolds alone has limited effect on cardiomyocyte maturation.

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