Stem Cell Differentiation Model Research Articles

Accurate isoform quantification by joint short- and long-read RNA-sequencing.

Accurate quantification of transcript isoforms is crucial for understanding gene regulation, functional diversity, and cellular behavior. Existing RNA sequencing methods have significant limitations: short-read (SR) sequencing provides high depth but struggles with isoform deconvolution, whereas long-read (LR) sequencing offers isoform resolution at the cost of lower depth, higher noise, and technical biases. Addressing this gap, we introduce Multi-Platform Aggregation and Quantification of Transcripts (MPAQT), a generative model that combines the complementary strengths of different sequencing platforms to achieve state-of-the-art isoform-resolved transcript quantification, as demonstrated by extensive simulations and experimental benchmarks. By applying MPAQT to an in vitro model of human embryonic stem cell differentiation into cortical neurons, followed by machine learning-based modeling of transcript abundances, we show that untranslated regions (UTRs) are major determinants of isoform proportion and exon usage; this effect is mediated through isoform-specific sequence features embedded in UTRs, which likely interact with RNA-binding proteins that modulate mRNA stability. These findings highlight MPAQT's potential to enhance our understanding of transcriptomic complexity and underline the role of splicing-independent post-transcriptional mechanisms in shaping the isoform and exon usage landscape of the cell.

Approximate Bayesian computation for inferring Waddington landscapes fromsingle-cell data.

Single-cell technologies allow us to gain insights into cellular processes at unprecedented resolution. In stem cell and developmental biology snapshot data allow us to characterize how the transcriptional states of cells change between successive cell types. Here, we show how approximate Bayesian computation (ABC) can be employed to calibrate mathematical models against single-cell data. In our simulation study, we demonstrate the pivotal role of the adequate choice of distance measures appropriate for single-cell data. We show that for good distance measures, notably optimal transport with the Sinkhorn divergence, we can infer parameters for mathematical models from simulated single-cell data. We show that the ABC posteriors can be used (i) to characterize parameter sensitivity and identify dependencies between different parameters and(ii) to construct representations of the Waddington or epigenetic landscape, which forms a popular and interpretable representation of the developmental dynamics. In summary, these results pave the way for fitting mechanistic models of stem cell differentiation to single-cell data.

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs

Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.

Dynamics of cell-type transition mediated by epigenetic modifications

Maintaining tissue homeostasis requires appropriate regulation of stem cell differentiation. The Waddington landscape posits that gene circuits in a cell form a potential landscape of different cell types, wherein cells follow attractors of the probability landscape to develop into distinct cell types. However, how adult stem cells achieve a delicate balance between self-renewal and differentiation remains unclear. We propose that random inheritance of epigenetic states plays a pivotal role in stem cell differentiation and present a hybrid model of stem cell differentiation induced by epigenetic modifications. Our comprehensive model integrates gene regulation networks, epigenetic state inheritance, and cell regeneration, encompassing multi-scale dynamics ranging from transcription regulation to cell population. Through model simulations, we demonstrate that random inheritance of epigenetic states during cell divisions can spontaneously induce cell differentiation, dedifferentiation, and transdifferentiation. Furthermore, we investigate the influences of interfering with epigenetic modifications and introducing additional transcription factors on the probabilities of dedifferentiation and transdifferentiation, revealing the underlying mechanism of cell reprogramming. This in silico model provides valuable insights into the intricate mechanism governing stem cell differentiation and cell reprogramming and offers a promising path to enhance the field of regenerative medicine.

γH2AX in mouse embryonic stem cells: Distribution during differentiation and following γ-irradiation

Phosphorylated histone H2AX (γH2AX) represents a sensitive molecular marker of DNA double-strand breaks (DSBs) and is implicated in stem cell biology. We established a model of mouse embryonic stem cell (mESC) differentiation and examined the dynamics of γH2AX foci during the process. Our results revealed high numbers of γH2AX foci in undifferentiated mESCs, decreasing as the cells differentiated towards the endothelial cell lineage. Notably, we observed two distinct patterns of γH2AX foci: the typical discrete γH2AX foci, which colocalize with the transcriptionally permissive chromatin mark H3K4me3, and the less well-characterized clustered γH2AX regions, which were only observed in intermediate progenitor cells. Next, we explored responses of mESCs to γ-radiation (137Cs). Following exposure to γ-radiation, mESCs showed a reduction in cell viability and increased γH2AX foci, indicative of radiosensitivity. Despite irradiation, surviving mESCs retained their differentiation potential. To further exemplify our findings, we investigated neural stem progenitor cells (NSPCs). Similar to mESCs, NSPCs displayed clustered γH2AX foci associated with progenitor cells and discrete γH2AX foci indicative of embryonic stem cells or differentiated cells. In conclusion, our findings demonstrate that γH2AX serves as a versatile marker of DSBs and may have a role as a biomarker in stem cell differentiation. The distinct patterns of γH2AX foci in differentiating mESCs and NSPCs provide valuable insights into DNA repair dynamics during differentiation, shedding light on the intricate balance between genomic integrity and cellular plasticity in stem cells. Finally, the clustered γH2AX foci observed in intermediate progenitor cells is an intriguing feature, requiring further exploration.

Single-cell transcriptomics identifies a WNT7A-FZD5 signaling axis that maintains fallopian tube stem cells in patient-derived organoids

The study of fallopian tube (FT) function in health and disease has been hampered by limited knowledge of FT stem cells and lack of invitro models of stem cell renewal and differentiation. Using optimized organoid culture conditions to address these limitations, we find that FT stem cell renewal is highly dependent on WNT/β-catenin signaling and engineer endogenous WNT/β-catenin signaling reporter organoids to biomark, isolate, and characterize these cells. Using functional approaches, as well as bulk and single-cell transcriptomics analyses, we show that an endogenous hormonally regulated WNT7A-FZD5 signaling axis is critical for stem cell renewal and that WNT/β-catenin pathway-activated cells form a distinct transcriptomic cluster of FT cells enriched in extracellular matrix (ECM) remodeling and integrin signaling pathways. Overall, we provide a deep characterization of FT stem cells and their molecular requirements for self-renewal, paving the way for mechanistic work investigating the role of stem cells in FT health and disease.

Self-Regulated Symmetry Breaking Model for Stem Cell Differentiation.

In conventional disorder-order phase transitions, a system shifts from a highly symmetric state, where all states are equally accessible (disorder) to a less symmetric state with a limited number of available states (order). This transition may occur by varying a control parameter that represents the intrinsic noise of the system. It has been suggested that stem cell differentiation can be considered as a sequence of such symmetry-breaking events. Pluripotent stem cells, with their capacity to develop into any specialized cell type, are considered highly symmetric systems. In contrast, differentiated cells have lower symmetry, as they can only carry out a limited number of functions. For this hypothesis to be valid, differentiation should emerge collectively in stem cell populations. Additionally, such populations must have the ability to self-regulate intrinsic noise and navigate through a critical point where spontaneous symmetry breaking (differentiation) occurs. This study presents a mean-field model for stem cell populations that considers the interplay of cell-cell cooperativity, cell-to-cell variability, and finite-size effects. By introducing a feedback mechanism to control intrinsic noise, the model can self-tune through different bifurcation points, facilitating spontaneous symmetry breaking. Standard stability analysis showed that the system can potentially differentiate into several cell types mathematically expressed as stable nodes and limit cycles. The existence of a Hopf bifurcation in our model is discussed in light of stem cell differentiation.

Estimation of non-equilibrium transition rate from gene expression data.

The dynamical properties of many complex physical and biological systems can be quantified from the energy landscape theory. Previous approaches focused on estimating the transition rate from landscape reconstruction based on data. However, for general non-equilibrium systems (such as gene regulatory systems), both the energy landscape and the probability flux are important to determine the transition rate between attractors. In this work, we proposed a data-driven approach to estimate non-equilibrium transition rate, which combines the kernel density estimation and non-equilibrium transition rate theory. Our approach shows superior performance in estimating transition rate from data, compared with previous methods, due to the introduction of a nonparametric density estimation method and the new saddle point by considering the effects of flux. We demonstrate the practical validity of our approach by applying it to a simplified cell fate decision model and a high-dimensional stem cell differentiation model. Our approach can be applied to other biological and physical systems.

Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR.

Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were validated by RT-qPCR. PPP6R1, CCDC97, and then either ACTB or EPHA2 demonstrated the most stable mRNA levels. Normalizing target gene Cq data with at least three of these RGs simultaneously, as per MIQE guidelines (PPP6R1 and CCDC97 with either ACTB or EPHA2), resulted in congruent conclusions. FABP5 expression was increased in ADs (5.99 and 8.00 fold, p = 0.00002 and p = 0.0003) and in OBs (5.18 and 5.91 fold, p = 0.0011 and p = 0.0023) relative to ADSCs. RUNX2 expression was slightly higher in ADs relative to ADSCs (1.97 and 2.65 fold, p = 0.04 and p = 0.01), but not in OBs (0.9 and 1.03 fold, p = 0.58 and p = 0.91).

Human Embryonic Stem Cells as a Model for Hematopoietic Stem Cell Differentiation and Viral Infection.

Pluripotent human embryonic stem cell (hESC) lines are a valuable in vitro tool to differentiate specific cell lineages, including cells from all three germ layers, i.e., neuronal cells, myocytes, and hematopoietic cells, including progenitors (described here), lymphoid cells, and myeloid cells. However, dramatically different cell subtypes and functional properties of specific cells can arise depending on the differentiation technique used. We previously optimized hematopoietic stem cell differentiation from two different NIH-approved hESC lines to generate CD34+ hematopoietic progenitor cells (HPCs). Infection of these HPCs with a common herpesvirus (human cytomegalovirus) results in maintenance of viral latency, capability of viral reactivation, recapitulation of viral mutant phenotypes, and virus-induced myelosuppression of hematopoietic differentiation. However, different HPC subpopulations support different viral latency and reactivation phenotypes, and different hESC-to-HPC differentiation methods alter the ratio of stem cell subsets. In addition, differences in differentiation methods are dependent on both protocol/reagents and user techniques. Here, we report a simplified and optimized method to generate large numbers of CD34+ HPCs with consistent phenotypes and demonstrate a comparison of several common methods that can be used to control the ratio of available HPC subpopulations. A key aspect of this approach is that we achieve consistency in differentiation across users in different laboratories and, importantly, among newly trained individuals. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance of human embryonic stem cells (hESCs) Basic Protocol 2: Differentiation of hESCs to hematopoietic progenitor cells (HPCs) Basic Protocol 3: Downstream functional differentiation of hESC-derived HPCs to mature lineages Support Protocol 1: Freezing and testing frozen batches of hESCs Support Protocol 2: Counting hESCs Support Protocol 3: Phenotyping by flow cytometry.

Exploring the neurogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins βIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.

A causal association of ANKRD37 with human hippocampal volume.

Human hippocampal volume has been separately associated with single nucleotide polymorphisms (SNPs), DNA methylation and gene expression, but their causal relationships remain largely unknown. Here, we aimed at identifying the causal relationships of SNPs, DNA methylation, and gene expression that are associated with hippocampal volume by integrating cross-omics analyses with genome editing, overexpression and causality inference. Based on structural neuroimaging data and blood-derived genome, transcriptome and methylome data, we prioritized a possibly causal association across multiple molecular phenotypes: rs1053218 mutation leads to cg26741686 hypermethylation, thus leads to overactivation of the associated ANKRD37 gene expression in blood, a gene involving hypoxia, which may result in the reduction of human hippocampal volume. The possibly causal relationships from rs1053218 to cg26741686 methylation to ANKRD37 expression obtained from peripheral blood were replicated in human hippocampal tissue. To confirm causality, we performed CRISPR-based genome and epigenome-editing of rs1053218 homologous alleles and cg26741686 methylation in mouse neural stem cell differentiation models, and overexpressed ANKRD37 in mouse hippocampus. These in-vitro and in-vivo experiments confirmed that rs1053218 mutation caused cg26741686 hypermethylation and ANKRD37 overexpression, and cg26741686 hypermethylation favored ANKRD37 overexpression, and ANKRD37 overexpression reduced hippocampal volume. The pairwise relationships of rs1053218 with hippocampal volume, rs1053218 with cg26741686 methylation, cg26741686 methylation with ANKRD37 expression, and ANKRD37 expression with hippocampal volume could be replicated in an independent healthy young (n = 443) dataset and observed in elderly people (n = 194), and were more significant in patients with late-onset Alzheimer's disease (n = 76). This study revealed a novel causal molecular association mechanism of ANKRD37 with human hippocampal volume, which may facilitate the design of prevention and treatment strategies for hippocampal impairment.

Abstract P3089: Etv2 Functions As A Pioneer Transcription Factor To Regulate Endothelial Lineage Development And Reprogramming

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and thus relevant to the treatment of ischemic heart disease. The ETS-related transcription factor 2 (ETV2) is a transcription factor that is both necessary and sufficient for the development of endothelial and blood lineages. However, the molecular mechanism by which ETV2 promotes endothelial and blood development is not known and warrants further investigation. Here, we report that ETV2 functions as a novel pioneer transcription factor that relaxes closed chromatin and regulates endothelial development. Pioneer factors are a subset of transcription factors that bind closed chromatin and promote chromatin relaxation in order to drive the development and reprogramming of cell lineages. By comparing embryonic stem cell differentiation and fibroblast reprogramming models with single cell RNA-seq, ATAC-seq, ChIP-seq and in-vitro nucleosomal binding techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and function as a pioneer factor independent of the cellular context. We also determined that ETV2 executed a pioneering role by recruiting and directly interacting with the ATP-dependent chromatin remodeling enzyme BRG1 to remodel chromatin around endothelial genes to help maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results that define a novel pioneer factor and epigenetic regulatory pathways that govern the specification and differentiation of endothelial progenitor cells, will serve as a platform for targeted therapies to promote cardiovascular regeneration.

Environmentally relevant exposure to TBBPA and its analogues may not drastically affect human early cardiac development

Tetrabromobisphenol A (TBBPA) and its substitutes and derivatives have been widely used as halogenated flame retardants (HFRs), in the past few decades. As a consequence, these compounds are frequently detected in the environment, as well as human bodily fluids, especially umbilical cord blood and breast milk. This has raised awareness of their potential risks to fetuses and infants. In this study, we employed human embryonic stem cell differentiation models to assess the potential developmental toxicity of six TBBPA-like compounds, at human relevant nanomolar concentrations. To mimic early embryonic development, we utilized embryoid body-based 3D differentiation in presence of the six HFRs. Transcriptomics data showed that HFR exposure over 16 days of differentiation only interfered with the expression of a few genes, indicating those six HFRs may not have specific tissue/organ targets during embryonic development. Nevertheless, further analyses revealed that some cardiac-related genes were dysregulated. Since the heart is also the first organ to develop, we employed a cardiac differentiation model to analyze the six HFRs’ potential developmental toxicity in more depth. Overall, HFRs of interest did not significantly disturb the canonical WNT pathway, which is an essential signal transduction pathway for cardiac development. In addition, the six HFRs showed only mild changes in gene expression levels for cardiomyocyte markers, such as NKX2.5, MYH7, and MYL4, as well as a significant down-regulation of some but not all the epicardial and smooth muscle cell markers selected. Taken together, our results show that the six studied HFRs, at human relevant concentrations, may impose negligible effects on embryogenesis and heart development. Nevertheless, higher exposure doses might affect the early stages of heart development.

ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage.

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and is thus relevant to the treatment of ischaemic diseases, injury-induced regeneration and solid tumour growth. Previously, we demonstrated that ETV2 is an essential transcription factor for the development of cardiac, endothelial and haematopoietic lineages. Here we report that ETV2 functions as a pioneer factor that relaxes closed chromatin and regulates endothelial development. By comparing engineered embryonic stem cell differentiation and reprogramming models with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. BRG1 recruitment remodelled chromatin around endothelial genes and helped to maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular diseases and solid tumours.

Screening for modulators of the cellular composition of gut epithelia via organoid models of intestinal stem cell differentiation

The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in wild-type mice. Physiological organoid models of the differentiation of intestinal stem cells could find broader utility for the screening of biological targets and small molecules that can modulate the composition and function of other barrier epithelia.

Endoderm and mesoderm derivatives in embryonic stem cell differentiation and their use in developmental toxicity testing

Embryonic stem cell differentiation models have increasingly been applied in non-animal test systems for developmental toxicity. After the initial focus on cardiac differentiation, attention has also included an array of neuro-ectodermal differentiation routes. Alternative differentiation routes in the mesodermal and endodermal germ lines have received less attention. This review provides an inventory of achievements in the latter areas of embryonic stem cell differentiation, with a view to possibilities for their use in non-animal test systems in developmental toxicology. This includes murine and human stem cell differentiation models, and also gains information from the field of stem cell use in regenerative medicine. Endodermal stem cell derivatives produced in vitro include hepatocytes, pancreatic cells, lung epithelium, and intestinal epithelium, and mesodermal derivatives include cardiac muscle, osteogenic, vascular and hemopoietic cells. This inventory provides an overview of studies on the different cell types together with biomarkers and culture conditions that stimulate these differentiation routes from embryonic stem cells. These models may be used to expand the spectrum of embryonic stem cell based new approach methodologies in non-animal developmental toxicity testing.

Investigating developmental effects of PFAS using a 3D human induced pluripotent stem cell differentiation model

Investigating developmental effects of PFAS using a 3D human induced pluripotent stem cell differentiation model

Abstract 185: Monitoring of stem cell differentiation to mature hepatocytes with a machine learning-based AI model

Abstract Human embryonic stem cells (hESCs) and pluripotent stem cells (iPSCs)-based disease modelings are potential platforms for cancer research and development of new cancer therapeutics. Differentiation of stem cells is an essential step for those disease models. For tissue-specific differentiation, hESCs or iPSCs are cultured in specific receipts of induction and differentiation media for developing different types of tissue cells such as muscle, skin, and liver providing further study or clinical applications. During lineage differentiation, researcher needs to closely monitor stem cell differentiation to be on track via checking cell morphological changes under microscope since this procedure has high probability of failed differentiation results (e.g., no differentiation or differentiating unwanted tissue types). However, monitoring via microscopy is labor-intensive and time-consuming, and also has high inter-observer variation issues. Therefore, it significantly impedes the progression of stem cell research and clinical applications nowadays. In recent years, machine learning has shown promising results in many applications of artificial intelligence (AI) in different fields, especially computer vision and image analysis. AI-based computational tool will bring benefits like high-throughput, high accuracy, and reproductivity in many medical applications. In stem cell culture and differentiation, we believe that applying this new technology will help researcher detect abnormal stem cell differentiation at the early stage via microscopy to save time, labor, and cost for the study and aggregate reproducible data along the process. To this end, we developed a machine learning-based AI model to assist in monitoring morphological changes of hESCs culture in bright-field microscopy images obtained from different differentiation stages to mature hepatocytes. We conducted a pilot study to train an AI model estimating efficiency of stem cell differentiation at Hepatic Progenitor Cell (HPC) stage, which is a critical checkpoint for hepatocyte differentiation. To prepare datasets for training, experienced researchers annotated the morphology of HPC in hundreds of microscope images and determined a differentiation result (success/fail) for every image. During the model training, the initial model was first trained by a training dataset consisting of 341 success and 366 fail HPC results. Subsequently, a smaller separate dataset comprising of 86 success and 51 fail HPC results was then used for cross-validation. Finally, the test set containing 64 success and 29 fail HPC results was used to evaluate the AI model performance. In result, the AI model presented an excellent performance (accuracy= 0.978 and F1 score= 0.975). Our study suggests a potential application of AI-assisted monitoring model for stem cell culture and differentiation in the future. Citation Format: Wei-Lei Yang, Zijun Huo, ShihYu Chen, Dandan Zhu, Tien-Jen Liu, Dung-Fang Lee. Monitoring of stem cell differentiation to mature hepatocytes with a machine learning-based AI model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 185.

SORBS2 is a genetic factor contributing to cardiac malformation of 4q deletion syndrome patients.

Chromosome 4q deletion is one of the most frequently detected genomic imbalance events in congenital heart disease (CHD) patients. However, a portion of CHD-associated 4q deletions without known CHD genes suggests unknown CHD genes within these intervals. Here, we have shown that knockdown of SORBS2, a 4q interval gene, disrupted sarcomeric integrity of cardiomyocytes and caused reduced cardiomyocyte number in human embryonic stem cell differentiation model. Molecular analyses revealed decreased expression of second heart field (SHF) marker genes and impaired NOTCH and SHH signaling in SORBS2-knockdown cells. Exogenous SHH rescued SORBS2 knockdown-induced cardiomyocyte differentiation defects. Sorbs2-/- mouse mutants had atrial septal hypoplasia/aplasia or double atrial septum (DAS) derived from impaired posterior SHF with a similar expression alteration. Rare SORBS2 variants were significantly enriched in a cohort of 300 CHD patients. Our findings indicate that SORBS2 is a regulator of SHF development and its variants contribute to CHD pathogenesis. The presence of DAS in Sorbs2-/- hearts reveals the first molecular etiology of this rare anomaly linked to paradoxical thromboembolism.