Circular RNA circZFPM2 regulates cardiomyocyte hypertrophy and survival.

Hypertrophic cardiomyopathy (HCM) constitutes the most common genetic cardiac disorder. However, current pharmacotherapeutics are mainly symptomatic and only partially address underlying molecular mechanisms. Circular RNAs (circRNAs) are a recently discovered class of non-coding RNAs and emerged as specific and powerful regulators of cellular functions. By performing global circRNA-specific next generation sequencing in cardiac tissue of patients with hypertrophic cardiomyopathy compared to healthy donors, we identified circZFPM2 (hsa_circ_0003380). CircZFPM2, which derives from the ZFPM2 gene locus, is a highly conserved regulatory circRNA that is strongly induced in HCM tissue. In vitro loss-of-function experiments were performed in neonatal rat cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and HCM-patient-derived hiPSC-CMs. A knockdown of circZFPM2 was found to induce cardiomyocyte hypertrophy and compromise mitochondrial respiration, leading to an increased production of reactive oxygen species and apoptosis. In contrast, delivery of recombinant circZFPM2, packaged in lipid-nanoparticles or using AAV-based overexpression, rescued cardiomyocyte hypertrophic gene expression and promoted cell survival. Additionally, HCM-derived cardiac organoids exhibited improved contractility upon CM-specific overexpression of circZFPM2. Multi-Omics analysis further promoted our hypothesis, showing beneficial effects of circZFPM2 on cardiac contractility and mitochondrial function. Collectively, our data highlight that circZFPM2 serves as a promising target for the treatment of cardiac hypertrophy including HCM.

Developmental Toxicity Studies: The Path towards Humanized 3D Stem Cell-Based Models.

Today, it is recognized that medicines will eventually be needed during pregnancy to help prevent to, ameliorate or treat an illness, either due to gestation-related medical conditions or pre-existing diseases. Adding to that, the rate of drug prescription to pregnant women has increased over the past few years, in accordance with the increasing trend to postpone childbirth to a later age. However, in spite of these trends, information regarding teratogenic risk in humans is often missing for most of the purchased drugs. So far, animal models have been the gold standard to obtain teratogenic data, but inter-species differences have limited the suitability of those models to predict human-specific outcomes, contributing to misidentified human teratogenicity. Therefore, the development of physiologically relevant in vitro humanized models can be the key to surpassing this limitation. In this context, this review describes the pathway towards the introduction of human pluripotent stem cell-derived models in developmental toxicity studies. Moreover, as an illustration of their relevance, a particular emphasis will be placed on those models that recapitulate two very important early developmental stages, namely gastrulation and cardiac specification.

Development of heart organoid cryopreservation method through Fe3 O4 nanoparticles based nanowarming system.

Beyond single cell two-dimensional (2D) culture, research on organoids that can mimic human organs is rapidly developing. However, there are still problems in commercialization and joint research using organoids due to the lack of technology to safely store organoids. Since organoids are 3D complex structures with a certain size (0.1-5 mm) beyond the size of cells, the conventional cell-level cryopreservation method using cryoprotectant (CPA) cannot overcome the damage caused by volume change due to osmotic pressure difference and ice nucleation. Herein, we attempted to solve such limitations by applying a nanowarming system using CPA with high cell permeability and Fe3 O4 nanoparticles. By performing beat rate measurement, histological analysis, contractility analysis, and multi-electrode array, it was verified that the developed method could significantly improve functional recovery and survival of heart organoids after freezing and thawing. In this study, we demonstrated a successful organoid cryopreservation method based on a Fe3 O4 nanowarming system. The developed technology will provide clues to the field of tissue cryopreservation and spur the application of organoids.

Polyoxyethylene tallow amine and glyphosate exert different developmental toxicities on human pluripotent stem cells-derived heart organoid model.

The early stage of heart development is highly susceptible to various environmental factors. While the use of animal models has aided in identifying numerous environmental risk factors, the variability between species and the low throughput limit their translational potential. Recently, a type of self-assembling cardiac structures, known as human heart organoids (hHOs), exhibits a remarkable biological consistency with human heart. However, the feasibility of hHOs for assessing cardiac developmental risk factors remains unexplored. Here, we focused on the cardiac developmental effects of core components of Glyphosate-based herbicides (GBHs), the most widely used herbicides, to evaluate the reliability of hHOs for the prediction of possible cardiogenesis toxicity. GBHs have been proven toxic to cardiac development based on multiple animal models, with the mechanism remaining unknown. We found that polyoxyethylene tallow amine (POEA), the most common surfactant in GBHs formulations, played a dominant role in GBHs' heart developmental toxicity. Though there were a few differences in transcriptive features, hHOs exposed to sole POEA and combined POEA and Glyphosate would suffer from both disruption of heart contraction and disturbance of commitment in cardiomyocyte isoforms. By contrast, Glyphosate only caused mild epicardial hyperplasia. This study not only sheds light on the toxic mechanism of GBHs, but also serves as a methodological demonstration, showcasing its effectiveness in recognizing and evaluating environmental risk factors, and deciphering toxic mechanisms.

Longitudinal morphological and functional characterization of human heart organoids using optical coherence tomography.

Organoids play an increasingly important role as in vitro models for studying organ development, disease mechanisms, and drug discovery. Organoids are self-organizing, organ-like three-dimensional (3D) cell cultures developing organ-specific cell types and functions. Recently, three groups independently developed self-assembling human heart organoids (hHOs) from human pluripotent stem cells (hPSCs). In this study, we utilized a customized spectral-domain optical coherence tomography (SD-OCT) system to characterize the growth of hHOs. Development of chamber structures and beating patterns of the hHOs were observed via OCT and calcium imaging. We demonstrated the capability of OCT to produce 3D images in a fast, label-free, and non-destructive manner. The hHOs formed cavities of various sizes, and complex interconnections were observed as early as on day 4 of differentiation. The hHOs models and the OCT imaging system showed promising insights as an in vitro platform for investigating heart development and disease mechanisms.

Modeling the Effects of Maternal Diabetes on the Developing Human Heart Using Pluripotent Stem Cell-Derived Heart Organoids.

Congenital heart defects (CHD) constitute the most common type of birth defect in humans. Maternal diabetes during the first trimester of pregnancy (pregestational diabetes, or PGD) is one of the most prominent factors contributing to CHD, and is present in a significant population of female patients with diabetes in reproductive age. PGD is challenging to manage clinically due to the extreme sensitivity of the developing embryo to glucose oscillations, and constitutes a critical health problem for the mother and the fetus. The prevalence of PGD-induced CHD is increasing due to the ongoing diabetes epidemic. While studies using animal models and cells in culture have demonstrated that PGD alters critical cellular and developmental processes, the mechanisms remain obscure, and it is unclear to what extent these models recapitulate PGD-induced CHD in humans. Clinical practice precludes direct studies in developing human embryos, further highlighting the need for physiologically relevant models. To bypass many of these technical and ethical limitations, we describe here a human pluripotent stem cell (hPSC)-based method to generate developmentally relevant self-organizing human heart organoids. By using glucose and insulin to mimic the diabetic environment that the embryo faces in PGD, this system allows modeling critical features of PGD in a human system with relevant physiology, structure, and cell types. The protocol starts with the generation of hPSC-derived embryoid bodies in a 96-well plate, followed by a small molecule-based three-step Wnt activation/inhibition/activation strategy. Organoids are then differentiated under healthy (normal insulin and glucose) and diabetic conditions (high insulin and glucose) over time, allowing for the study of the effects of pregestational diabetes on the developing human heart. We also provide an immunofluorescence protocol for comparing, characterizing, and analyzing the differences between the healthy and diabetic organoids, and comment on additional steps for preparing the organoids for analysis by other techniques after differentiation. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of hPSC-derived embryoid bodies Basic Protocol 2: Differentiation of EBs into heart organoids under healthy and diabetes-like conditions Basic Protocol 3: Immunofluorescence and organoid preparation for other assays.