However, treatment with RepSox or DM induced either no Ca2+ oscillation or only Ca2+ oscillation with very small peak amplitudes in 3D and 2D cultures at day 7 and 14. Open in a separate window Figure 6 Effect of BMP signaling inhibition and ALK5 inhibitor on CPC differentiation in 3D vs 2D culture. with increased number of cardiac Troponin T (cTnT)-positive cells and synchronized intracellular Ca2+ oscillation. In addition, we studied if 3D nanofiber culture can be used as an in vitro model for compound screening by testing a number of other differentiation factors including a ALK5 inhibitor and inhibitors of BMP signaling. This work highlights the importance of using a more relevant in vitro model and measuring not only the expression of marker proteins but also the functional readout in a screen in order to identify the best compounds and to investigate the resulting biology. Many examples across different cell types have shown that cell morphology, function, and fate are influenced by the physical interactions of cells with the extracellular matrix (ECM)12C16. During the past 20?years, increased efforts have been made to culture cells in a more in vivo-like environment by using three-dimensional (3D) culture systems with Mecarbinate relevant matrix components. Numerous studies have exhibited that in vitro cellular models with cells grown in 3D culture, which structurally mimic the architecture of the ECM of the native tissue, have higher predictivity in in vitro models than 2D culture models for studying cell biology and disease pathophysiology, and for identifying therapeutic brokers17C19. For example, HepG2 liver cancer cells in 3D spheroids produce their own ECM and are highly organized and tissue-like20, fibroblasts cultured with collagen gels and fibronectin-containing matrices that mimic in vivo 3D environment exert different drug responsiveness compared to cells growing in 2D cultures21. However, using 3D culture models, such as spheroids, organoids and organ-on-a-chip in drug discovery for screening large Mecarbinate numbers of compounds (e.g. in a phenotypic screening campaign) can still be very challenging as these more complex assays are difficult to adapt to plate-based medium-to-high throughput screening and automation. In recent years, Mecarbinate a number of plate-based 3D culture models, such as low attachment and hanging drop plates for spheroids, plates with nanofibrous scaffolds composed of electrospun synthetic nanofibers, and plates coated with hydrogels, have become commercially available. Such 3D cultures have been investigated in different cellular models to improve physiological relevance, including human adipose-derived stem cells15, rat hippocampal embryonic neurons22, bovine pulmonary artery easy muscle cells23, and hepatocytes24,25. Adult CMs in vivo have an elongated morphology, but, CMs cultured in standard 2D condition do not show elongated morphology. Numerous studies have shown that aligned nanofiber scaffolds guide CM cell alignment along the Rabbit polyclonal to RAB18 direction of fiber orientation, promote adaptation of an elongated CM morphology, and improve CM function and maturation when CMs are cultured in 3D aligned nanofiber scaffolds26C32. The potential of engineered cardiac tissue like constructs obtained by seeding CMs on aligned nanofibers or into a 3D fibrin scaffold for treating myocardial infarction has been exhibited8,32. In addition, several groups also reported that nanofiber scaffolds enhance cardiac differentiation of stem cells or progenitor cells33C37. Thus, there is a clear rationale to investigate if nanofiber scaffolds can improve iPCS-CPCs differentiation into CMs to provide a more effective and relevant model or phenotypic assay, particularly if scaleable for medium-to-high throughput drug discovery. For this study, 384-well 3D nanofiber plates with aligned polycaprolactone (PCL) nanofiber scaffolds, which structurally mimic the architecture of CMs in the heart38, was chosen to investigate the effect of 3D scaffolds on human iPSC-CPC cardiac differentiation brought on by Wnt inhibition that is reported to promote iPSC-CPC cardiac differentiation. Protein and gene expression of CM and SMC markers, and intracellular Ca2+ oscillation were used for cardiac differentiation assessment. In addition, we studied if 3D nanofiber culture can be used as an in vitro model for compound screening by testing alternative molecules which have been shown to differentiate iPSC-CPCs in 2D culture. Results Wnt signaling inhibition induced differentiation of human iPSC-CPCs in 3D and 2D culture Human iPSC-CPCs were treated with 10?M XAV939, 1.1?M 53AH (a structurally diverse inhibitor of Wnt signalling), or DMSO control in triplicates. Cells were fixed at day 7 or day 14 of differentiation, then stained for cardiac Troponin T (cTnT) and easy muscle actin (SMA) for studying iPSC-CPC differentiation. These concentrations and time points were selected based on our previously obtained knowledge for the differentiation of CPCs to CMs and that described in the literature10,39. Physique?1 shows an outline of the human iPSC-CPC differentiation protocol, and double immunostaining with cTnT and SMA of cells at day 14 in 3D versus 2D culture treated with XAV939, 53AH or DMSO, as well as aligned fibers on 3D nanofiber Mecarbinate plates. Open in a separate window Physique 1 Schematic outline of the CPC differentiation protocol and immunofluorescence staining of CPC differentiation in 3D aligned nanofiber plates and.
However, treatment with RepSox or DM induced either no Ca2+ oscillation or only Ca2+ oscillation with very small peak amplitudes in 3D and 2D cultures at day 7 and 14