Human Induced Pluripotent Stem Cell-Derived Cardioids as a Model to Assess Oligonucleotide Delivery.
Oligonucleotide-based therapeutics represent a rapidly advancing class of drugs with significant potential for treating cardiovascular diseases; however, achieving efficient delivery to cardiac tissue remains a critical and unresolved challenge. A key obstacle is the limited availability of robust, physiologically relevant human in vitro models capable of supporting quantitative assessment of oligonucleotide cellular uptake and intracellular distribution. A detailed, step-by-step protocol is presented for generating self-organizing, 3D cardioids from human induced pluripotent stem cells (iPSCs) and applying them as a platform to evaluate the uptake of fluorescently labeled oligonucleotides. The protocol guides users through directed cardiac differentiation in suspension culture by temporally modulating Wnt/β-catenin signaling, enabling sequential specification of iPSCs through the mesoderm, cardiac mesoderm, and cardiomyocyte progenitor stages. Under these conditions, cells spontaneously self-assemble into beating, cavity-containing three-dimensional structures that express canonical cardiomyocyte markers. The resulting cardioids provide a scalable, experimentally tractable platform for imaging-based assessment of oligonucleotide uptake efficiency, supporting the development and optimization of delivery strategies for cardiac applications.