Abstract
Cardiac progenitor cells (CPCs), capable of differentiating into multiple cardiac cell types including cardiomyocytes (CMs), endothelial cells, and smooth muscle cells, are promising candidates for cardiac repair/regeneration. In vitro model systems where cells are grown in a more in vivo-like environment, such as 3D cultures, have been shown to be more predictive than 2D culture for studying cell biology and disease pathophysiology. In this report, we focused on using Wnt inhibitors to study the differentiation of human iPSC-CPCs under 2D or 3D culture conditions by measuring marker protein and gene expression as well as intracellular Ca2+ oscillation. Our results show that the 3D culture with aligned nanofiber scaffolds, mimicing the architecture of the extracellular matrix of the heart, improve the differentiation of iPSC-CPCs to functional cardiomyocytes induced by Wnt inhibition, as shown 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.
Highlights
Cardiac progenitor cells (CPCs), capable of differentiating into multiple cardiac cell types including cardiomyocytes (CMs), endothelial cells, and smooth muscle cells, are promising candidates for cardiac repair/regeneration
Numerous studies have demonstrated that in vitro cellular models with cells grown in 3D culture, which structurally mimic the architecture of the extracellular matrix (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 a gents[17,18,19]
Cells were fixed at day 7 or day 14 of differentiation, stained for cardiac Troponin T and smooth muscle actin for studying induced pluripotent stem cell (iPSC)-CPC differentiation
Summary
Cardiac progenitor cells (CPCs), capable of differentiating into multiple cardiac cell types including cardiomyocytes (CMs), endothelial cells, and smooth muscle cells, are promising candidates for cardiac repair/regeneration. Our results show that the 3D culture with aligned nanofiber scaffolds, mimicing the architecture of the extracellular matrix of the heart, improve the differentiation of iPSC-CPCs to functional cardiomyocytes induced by Wnt inhibition, as shown with increased number of cardiac Troponin T (cTnT)-positive cells and synchronized intracellular Ca2+ oscillation. 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 c ells[15], rat hippocampal embryonic neurons[22], bovine pulmonary artery smooth muscle cells[23], and hepatocytes[24,25]. 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, if scaleable for medium-to-high throughput drug discovery
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