Human Induced Pluripotent Stem Cell-Derived Cardioids as a Model to Assess Oligonucleotide Delivery

Summary

This protocol enables the generation of self-organizing cardiomyocyte-based cardioids from human induced pluripotent stem cells for microscopic assessment of fluorescently labeled oligonucleotide uptake.

Abstract

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.

Introduction

Oligonucleotide-based therapeutics, primarily antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), are rapidly emerging as a transformative and highly specific approach for the treatment of a broad range of diseases, including both hereditary and acquired conditions^1,2,3. These modalities enable direct modulation of gene expression at the mRNA level through sequence-specific binding to target transcripts, allowing gene silencing or splicing correction of disease-associated genes that are often inaccessible to conventional small-molecule therapies

Protocol

1. Culture and preparation of iPSCs for differentiation

CAUTION: Perform all open handling of cells and media in a certified Class II biosafety cabinet. Wear a lab coat and gloves. Decontaminate work surfaces and liquid waste with an appropriate disinfectant.

1. Maintain iPSCs.
   1. Coat culture plates with an appropriate basement-membrane matrix according to the manufacturer’s instructions.
   2. Feed iPSCs with a defined, feeder-free pluripotent stem cell medium and replace medium every 24 h.
   3. Maintain cultures below over-confluency and passage cells when they reach 70–80% conflu....

Results

Figure 1 summarizes the differentiation workflow and illustrates the characteristic morphological changes expected at each stage of the protocol, from iPSC aggregation to the formation of self-organizing cardioids. Suspension-based microwell aggregation provides a simple and standardized approach for the parallel generation of large numbers of size-controlled cardioids. Successful differentiation is characterized by the generation of uniformly sized aggregates, followed by the development of.......

Discussion

Oligonucleotide-based therapeutics have emerged as a powerful class of gene-targeting modalities, supported by decades of advances in oligonucleotide chemistry that have improved target specificity, metabolic stability, and immunogenic tolerability^7,8. Despite the increasing number of clinically approved RNA therapeutics, their broader application remains limited by inefficient intracellular delivery^7,8. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4', 6-diamidino-2'-phenylindole, dihydrochloride	Thermo Scientific	62248	DAPI
10× dry objective	Zeiss		EC Plan-Neofluar 10×/0.30
40× water objective	Zeiss		LD C-Apochromat 40×/1.10 Corr
63× oil-immersion objective	Zeiss		Plan-Apochromat 63×/1.40 Oil DIC M27
800-µm microwell aggregation plate	STEMCELL Technologies	34815	AggreWell 800
Andor Neo 5.5 sCMOS camera	Andor		Andor Neo 5.5
Antifade mounting medium	Vector Laboratories	H-1000-10	VECTASHIELD Antifade Mounting Medium
B27 supplement minus insulin, 50×	Gibco	A18956-01	B27 Minus Insulin 50×
B27 supplement with insulin, 50×	Gibco	17504-044	B27 Supplement 50×
Basal Medium	Gibco	21875-034	RPMI 1640
Basement-membrane matrix	Gibco / Corning	A14132-02 / 354230	Geltrex LDEV-Free / Matrigel LDEV-Free
brightfield microscope	EVOS		EVOS XL Core Imaging Microscope
Cell detachment solution	STEMCELL Technologies	07922	ACCUTASE
confocal microscope	ZEISS		ZEISS LSM 980 with Airyscan 2 Microscope
Defined feeder-free iPSC maintenance medium	STEMCELL Technologies	100-0276	mTeSR Plus
DMEM/F12	Gibco	31331-028	DMEM/F12
Non-treated 6-well plate	Avantor (VWR)	734-2777	Untreated 6-well Plate
Phalloidin	Thermo Scientific	A22287	Phalloidin Labeling Probes ALexa 647
Phosphate-buffered saline (1× PBS)	Gibco	14190-144	DPBS 1×
primary antibody c-MYBPC3	Santa Cruz	sc-137181	MYBPC3 Antibody (F-1)
ROCK inhibitor	STEMCELL Technologies	72304	ROCK Inhibitor (Y-27632)
Secondary antibody (green)	Thermo Scientific	A-11017	F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488
Washing medium supplement	Gibco	10828028	KnockOut Serum Replacement
WGA, Alexa Fluor™ 488 conjugate	Invitrogen	W11261	WGA
widefield microscope	Nikon		Nikon Eclipse Ti
WNT pathway inhibitor	STEMCELL Technologies	72552	IWP4
Wnt/β-catenin pathway activator	Sigma	SML1046	CHIR99021