Electrospinning is an effective platform technique

Electrospinning is an effective platform technique to produce anisotropic (aligned) fibrous scaffolds (Barnes et al., 2007), comparing to conventional methods in fabricating aligned substrates, such as soft lithography (Kim et al., 2010; McCain et al., 2014; Rao et al., 2013; Salick et al., 2014), photolithography, two-photon initiated polymerization (Ma et al., 2014), and micro-fluidics (Xiao et al., 2014). Importantly, the as-prepared electrospun scaffolds possess a fibrous structure resembling the native extracellular matrix (ECM), a high surface-to-volume ratio, and a tunable porosity (Li et al., 2002). Remarkably, those fibrous meshes can be easily fabricated with distinct anisotropy through the use of a non-conductive template, as the surface topography and architecture of the non-conductive template is readily transferrable (Senel Ayaz et al., 2014; Zhao et al., 2013). A recent study has shown that using a textile-templated electrospun aligned PU scaffold as the substrate, neonatal rat CMs displayed more stable and prolonged spontaneous syncytium, comparing with buy AL 8697 on tissue culture polystyrenes (TCPs) (Senel Ayaz et al., 2014). Based on these findings, we examined whether aligned electrospun fibrous scaffolds could induce the anisotropic cell alignment and improve the maturation of hPSC-CMs. To this end, we first prepared the aligned and isotropic (random) polycaprolactone (PCL) fibrous scaffolds, and confirmed the anisotropic and isotropic alignment of hPSC-CMs cultured on these substrates coated with Matrigel, including the use of TCPs as a control. We then evaluated the structural, molecular and functional properties of the cells after 2weeks culture on each type of substrates. Our results show that aligned electrospun fibrous scaffolds can induce the anisotropic cell alignment of hPSC-CMs but do not improve the maturation of hPSC-CMs.
Materials and methods
Results
Discussion Comparing with cells on the TCPs, hPSC-CMs grown on the aligned fibrous scaffolds had enhanced molecular maturation. For instance, genes encoding ion channel or calcium handling proteins, such as ATP2A2/SERCA2, CASQ2, HCN1, KCNJ2, and KCNA4, were universally increased in cells on aligned fibrous scaffolds. Particularly, CASQ2 and KCNJ2, encoding the most abundant, high-capacity but low-infinity Ca-binding protein calsequestrin that stores and buffers Ca in the sarcoplasmic reticulum (Liu et al., 2009) and potassium inwardly rectifying Kir2.1 channel, respectively, were also significantly upregulated when compared to the random. These upregulations are consistent with the reported observations for hPSC-CMs subjected to prolonged culture, electrical stimulation, mechanical stretch or 3-D tissue formation (Lieu et al., 2013; Lundy et al., 2013; Mihic et al., 2014; Tulloch et al., 2011; Zhang et al., 2013) which were shown to enhance CM maturation. A higher proportion of hPSC-CMs on aligned fibrous scaffolds had increased levels of sarcomeric striations than cells on TCPs, albeit the average length of sarcomeres was similar and the average cell size of hPSC-CMs from cultures on the scaffolds was significantly smaller. The observation of reduced cell area for cells on the fibrous scaffolds is consistent with the published literatures (Bashur et al., 2006; Han et al., 2013), where reduced sizes of human aortic endothelial cells and 3T3 fibroblasts were observed in cells on the random electrospun fibrous scaffolds, comparing with those on the natural protein-coated glass coverslips or TCPs. It was reported that for substrata with uniform ridges and grooves, cells tended to reduce their sizes when they were on surfaces with either over-narrowed or over-wide scales, because of the limited cell penetration into over-narrowed grooves, or the loss of the extra cell-substratum contact on surfaces with the over-wide grooves (Kim et al., 2010). We surmise that our submicron electrospun fibers, in a 3-D scaffolding environment, may confine the penetration of the cells therefore reduced the cell spatial organization. Future studies with an optimal fiber size/scaffold topology which can be modulated by the electrospinning parameters (Kuroda et al., 2014) may increase the cell spreading and improve cell maturation (Han et al., 2013). In addition, a stage-specific and dynamic design of the fiber size/scaffold topology may be more beneficial, as the size of hPSC-CMs constantly enlarges during maturing.