Nemaline myopathy is a rare genetic condition characterized by weakened muscles due to thread-like rods, called nemaline bodies, in muscle fibers. This condition varies in time of onset and severity. Although there is no cure, therapies and treatments are available to reduce symptoms. This iPSC line, NCHi023-A, was reprogrammed using Sendai virus from skin fibroblasts from a male patient with nemaline myopathy carrying a pathogenic heterozygous ACTA1 mutation. Characterization of this line was successful, with validation of cell identity, normal morphology and karyotype, positive expression of germ layer and pluripotency markers, and negative expression for mycoplasma and transgenes.

Introduction: Hypoplastic left heart syndrome (HLHS) is a rare congenital defect marked by the underdevelopment of the left-sided structures in the heart. Currently, available animal models mimicking left ventricular hypoplasia are constrained because they often fail to reproduce the full spectrum of structural and hemodynamic abnormalities. In this study, we aim to employ a self-organizing cardioid model derived from patient-specific induced pluripotent stem cells to investigate the molecular and cellular mechanisms that underlie ventricular malformation in HLHS. Approaches: We generated cardioids from induced pluripotent stem cell (iPSC) lines derived from three pairs of HLHS probands and their sex-matched unaffected family controls. We measured the overall size and recorded the onset of cavity formation in cardioids from both HLHS patients and healthy controls between day 2 and day 8. Cardioids collected at days 2, 5, 8, 10, 14, and 20 were subjected to bulk RNA sequencing. Additionally, spatial profiling of cryo-sectioned and embedded day 20 cardioids was performed using Visium spatial transcriptomics. Results: Cardioids derived from HLHS patients displayed a smaller overall size accompanied by a slower growth rate compared to healthy controls. HLHS patient-specific cardioids show delayed formation of chamber-like cavity than gender-matched family controls. Time-course transcriptomic profiling of cardioids confirms efficient cardiomyocyte differentiation, as demonstrated by abundant expression of cardiac lineage marker genes. Bulk RNA sequencing of day 2 cardioids reveals significant downregulation of NFATC4, a gene essential for cardiomyocyte proliferation, and FHL1, which is typically upregulated in patients with hypertrophic cardiomyopathy. Further analysis of day 10 cardioids shows notable downregulation in pathways associated with cardiac muscle contraction, cardiac chamber development, and ventricular cardiac muscle tissue development. Spatial transcriptomics performed on day 20 cardioids, along with supporting immunofluorescence data, demonstrate the presence of myocardial and endocardial layers in both HLHS patients and healthy controls. Enrichment analysis of biological pathways indicates that myocardial layer in HLHS-derived cardioids is less proliferative compared to healthy controls. Conclusions: HLHS patient-specific cardioids provide a unique in vitro model to dissect the cellular etiologies of HLHS in a 3D context.

Background: Down syndrome (DS) is the most common genetic abnormality caused by an extra copy of chromosome 21 with nearly half of these individuals having a congenital heart defect (CHD), specifically an atrioventricular septal defect (AVSD), as a co-occurring condition. Current paradigm holds that CHD occurrence is due to the gene dosage imbalance caused by the triplication of chromosome 21. While mouse models have been developed to study DS and recapitulate some of the cardiac phenotypes present in the clinic, patient-specific induced pluripotent stem cells (iPSCs) provide a patient-specific context to investigate cardiac cellular differentiation deficits. Objective: Determine cardiac cell lineage commitment defects in DS+AVSD iPSC-CM and assess ventricular cardiomyocyte characteristics and functions. Methods: iPSCs were generated from the proband and gender-matched family member followed by a Wnt-ON/Wnt-OFF cardiomyocyte differentiation protocol. Single-cell RNAseq was performed on differentiating iPSCs at Days 5 (cardiac mesoderm), Day 10 (cardiac progenitors), Day 14 (early cardiomyocytes) and Day 30 (cardiomyocytes) of the differentiation process. Bioinformatic analysis included differential gene expression, pathway analysis, pseudotime trajectory, and cell-cell communication inference. Day 30 iPSC-derived cardiomyocytes (iPSC-CMs) were further assessed with immunofluorescence for ventricular markers, extracellular flux assay for mitochondrial respiration, and calcium imaging for beat frequency and cell-cell coupling. Results: Day 5 pathway analysis in DS versus Control showed a global increase in pathways associated with muscle cell development and muscle contraction while a decrease in mesoderm formation, GPCR signaling, and axis specification pathways. Day 10 and 14 DS cell populations show an emergence of mitochondrial and splicing dysfunction pathways in cardiac progenitors while endoderm progenitors showed an upregulation in inflammatory and cytokine pathways. D30 iPSC-CMs show significant downregulation in cardiac conduction pathways evidenced in vitro by decreased localization of gap junction protein, connexin-43, to cell-cell boundaries and abnormal formation of excitation-contraction proteins. Day 30 iPSC-CMs also exhibit aberrant calcium handling and mitochondrial dysfunction. Conclusion: DS iPSC-CMs recapitulate cardiac phenotypes seen in DS patients and provide a cell-type specific context to DS-related congenital heart defects.

Bicuspid aortic valve is one of the most common congenital heart defects which have only two leaflets instead of three. NCHi024-A is an induced pluripotent stem cell (iPSC) line derived from peripheral blood mononuclear cells (PBMCs) of a male infant with a bicuspid aortic valve. The line exhibits normal iPSC morphology, normal karyotype, and donor-matched identity. Markers of undifferentiated iPSC and differentiation into ectoderm, mesoderm, and endoderm were validated through immunofluorescence staining. NCHi024-A was tested negative for transgenes and mycoplasma contamination. This iPSC line can be differentiated into various types of cells and used for studying aortic valve disease.

Single ventricle heart defects are the most severe forms of congenital heart defects and are classified based on the affected ventricles: hypoplastic left heart syndrome (HLHS) and hypoplastic right heart syndrome (HRHS). Cellular etiologies underlying differential left and right ventricular hypoplasia are unknown and remain intractable without an experimental model to probe common and divergent cellular etiologies between HLHS and HRHS. In this study, we leveraged patient-specific induced pluripotent stem cells (iPSCs) and single-cell transcriptomics to interrogate distinct etiologies of HLHS and HRHS. Both HLHS and HRHS iPSC-derived cardiomyocytes (iPSC-CMs) exhibit a reduced proliferation capacity compared to sex-matched family controls under both static and cyclic stretch conditions. Biological pathways related to cell cycle progression, DNA replication, and cell proliferation are downregulated in both HLHS and HRHS iPSC-CMs, suggesting an intrinsic cardiac proliferation deficiency. Single-cell transcriptomics indicate that differentiation of cardiac mesoderm towards second heart field (SHF) progenitors are compromised in both HLHS and HRHS. However, differentiation of epicardial progenitors is enhanced at the expense of SHF progenitors in HLHS, whereas first heart field (FHF) progenitors are prevalent in HRHS. Trajectory inference analysis uncovers distinct cell lineage determination pathways in FHF and SHF progenitors between HLHS and HRHS. Moreover, HLHS iPSC-CMs exhibit reduced mitochondrial activities whereas HRHS iPSC-CMs show enhanced mitochondrial respiration and ATP production compared to controls. In sum, these common and divergent cellular etiologies of HLHS and HRHS may underlie ventricular hypoplasia in the left side and right side of the heart.

HAND2 is a transcription factor that plays a vital role in the development of the heart, limbs, and pharyngeal arch. Functional defects in HAND2 have been shown to cause congenital malformations in the extremities of the body and the heart. NCHi025-A iPSC line was generated from a 1-year-old female with pulmonary stenosis and harbors a novel de novo heterozygous variant of uncertain significance within HAND2 (NM_021973.3; c.247delG; p.Val83CysfsTer16). This variant causes a frameshift and premature stop codon that is predicted to truncate the protein. NCHi025-A is a pluripotent stem cell line that can be leveraged to investigate HAND2-associated phenotypic development.

Pulmonary atresia with intact ventricular septum (PA/IVS) is a rare congenital heart defect that causes a significant decrease of blood outflow from the heart and is fatal if left untreated. iPSC line NCHi013-A was produced from peripheral blood mononuclear cells from a male child with PA/IVS using Sendai virus reprogramming. NCHi013-A displayed normal stem cell morphology, expressed markers for pluripotency, and presented ability to differentiate into cells of endoderm, ectoderm, and mesoderm lineages. The iPSC line also maintained normal karyotype, was validated for cell identity, and tested negative for transgenes and mycoplasma contamination.

Maternal diabetes is associated with a 4-fold increased risk of offspring developing congenital heart defects (CHDs). It remains unknown how maternal diabetes interferes with cardiac cell lineage determination and leads to malformations in the heart. In this study, we aim to elucidate the cellular mechanisms by which maternal hyperglycemia causes high risk of CHDs in newborns. Here we leverage an in vitro hyperglycemic model using human induced pluripotent stem cells (iPSCs), which could recapture cardiac differentiation and cell lineage commitment during embryonic heart development. We collected differentiating cells at D5 (cardiac mesoderm), D10 (cardiac progenitors), and D14 (early cardiomyocytes) during cardiac differentiation under normal and hyperglycemic conditions, and performed single-cell transcriptomic analysis. We found that hyperglycemia significantly impedes cardiac differentiation of human iPSCs as robust cardiac differentiation is rarely observed in multiple iPSC lines (n=10) under hyperglycemia. At the cellular level, hyperglycemia interferes with cardiac differentiation of human iPSCs in response to WNT signaling activation, which is manifested by reduced number of cardiac mesoderm (MESP1+ PDGFRA+) cells at D5 of differentiation. In contrast, neural differentiation is enhanced under hyperglycemia, with a high proportion of neural cell lineage (SOX2+ PAX6+). At D10, differentiated neural cell lineage dominates the cell population under hyperglycemia at the expense of cardiac progenitors and early cardiomyocytes. At D14, the prevalence of neural lineage persist whereas early cardiomyocytes only accounts for a small portion of cell population under hyperglycemia. Moreover, we treated D30 iPSC-derived cardiomyocytes (iPSC-CMs) with high glucose concentration (25 mM) for 7 days and found that iPSC-CMs show reduced mitochondria respiration and ATP production, but elevated apoptosis and reactive oxygen species (ROS) generation. Together, our data suggest that maternal hyperglycemia could interrupt human embryonic heart morphogenesis through overriding WNT-medicated cardiac differentiation and promoting neural cell lineage determination by default.

Congenital single-ventricle heart defects (SVHDs) are life-threatening conditions in newborns, where one of the heart's ventricular chambers is severely underdeveloped. While medical advancements have improved survival rates, many SVHD patients still suffer lifelong cardiac complications and comorbidities. Clinical diagnosis of fetal SVHD occurs at around 20 weeks of gestation through echocardiogram. In this study, we aimed to identify novel maternal blood biomarkers that could signal SVHD development in fetuses. The study's premise is based on the limited regenerative capacity of adult human cardiomyocytes, like those in pregnant mothers, compared to the extensive proliferation seen in fetal cardiomyocytes that form the functional heart. We hypothesized small signaling molecules secreted by proliferating fetal cardiomyocytes enter the maternal circulatory system and could indicate abnormal ventricular growth in SVHD-affected fetuses. Both clinical and in vitro approaches were employed to identify potential cell-free miRNAs released by the developing fetal heart into maternal circulation. Blood plasma samples from pregnant women carrying healthy and SVHD-affected fetuses were screened for differentially expressed cell-free miRNAs and were compared with those from the supernatants of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) grown from healthy and SVHD donors. This comparative approach led to the identification of an miRNA panel (miR-487b, miR-433, mi-134, and miR-889) that were elevated in pregnant women carrying SVHD-affected fetuses and in proliferating iPSC-CMs from SVHD patients compared to healthy controls (p.adj<0.05). From this panel, forced-expression of miR-487b and mir-134 modulated the proliferation profiles of human cardiomyocytes in vitro . Results from this study establish these circulating cell-free miRNAs in maternal blood as potential non-invasive biomarkers for predicting abnormal fetal cardiac development leading to SVHDs. These findings could potentially revolutionize SVHD screening as an alternative diagnostic tool to echocardiogram.

Pulmonary atresia with intact ventricular septum (PA-IVS) is a rare congenital heart defect characterized by underdeveloped pulmonary valve and right ventricular hypoplasia. Neonates undergoing surgery to open pulmonary valve have a range of post-surgical ventricular recovery: single-ventricle (1v) palliation, one-and-half ventricle (1.5v) palliation, and bi-ventricular (2v) repair. PA-IVS-1.5v typically requires surgical intervention to install cavopulmonary shunt and entails partial right ventricle recovery. NCHi016-A is an iPSC line derived from a 5-year-old female with PA-IVS-1.5v using Sendai Virus reprogramming. This iPSC line shows typical iPSC morphology, has normal karyotype, expresses pluripotency markers, and has potential to differentiate into three germ layers.

Truncus arteriosus (TA) is a congenital heart defect where one main blood vessel emerges from the heart, instead of individual aorta and pulmonary artreries. Peripheral mononuclear cells (PBMCs) of a male infant with TA were reporogrammed using Sendai virus. The resultant iPSC line (NCHi015-A) displayed normal colony formation, expressed pluripotency markers, and differentiated into cells from three germ layers. NCHi015-A was matched to the patient’s genetic profile, had normal karyotype, retained genetic variants in KMT2D and NOTCH1, and tested negative for reprogramming transgene. This iPSC line can be used for studying congenital heart defects associated with genetic variants in KMT2D and NOTCH1.

Background: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. Methods: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. Results: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. Conclusions: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.

Pulmonary atresia with intact ventricular septum (PA-IVS) is a rare (4-8 per 100,000 live births) type of hypoplastic right heart syndrome (HRHS) where the right-sided structures in the heart are malformed. In PA-IVS, the pulmonary valve that acts to regulate the unidirectional flow of blood from the right ventricle (RV) to the lungs does not open, resulting in no connection between the RV and the pulmonary arteries. After surgical or catheter-based intervention, PA-IVS patients have clinical outcomes that range from single-ventricle palliation to a biventricular repair. Mechanisms underlying the spectrum of RV hypoplasia in PA-IVS are difficult to fully ascribe to an atretic pulmonary valve. There are no reliable animal models available for studying disease mechanisms of PA-IVS. In this study, we leverage patient-specific induced pluripotent stem cells (iPSCs) and single-cell RNA sequencing to elucidate cellular etiologies of ventricular hypoplasia in PA-IVS. PA-IVS iPSC-derived cardiomyocytes are less proliferative compared to controls under both static and cyclic stretch, suggesting that genetic factors may contribute to a spectrum of ventricular hypoplasia in PA-IVS. Single-cell transcriptomic analysis reveals that cell lineage determination towards second heart progenitors is suppressed, with enhanced differentiation into first heart field and epicardial lineages during cardiac differentiation. Additionally, biological pathways associated with cell proliferation and cell cycle progression are downregulated whereas mitochondrial activities are elevated in early cardiomyocytes originated from PA-IVS patients. In conclusion, abnormal cell lineage differentiation and cardiomyocyte proliferation may underlie the cellular and developmental etiologies of ventricular hypoplasia in HRHS.

NOTCH1 signaling is crucial for cardiovascular development. Numerous studies have identified heterozygous NOTCH1 loss of function and missense variants associated with a spectrum of congenital heart diseases (CHD). We generated induced pluripotent stem cells (iPSC) from a healthy individual to develop a model for NOTCH1+/- iPSC to study the molecular pathogenesis of CHD. NOTCH1+/-iPSC (NCHi014-A) have normal morphology and karyotype, are identical to the parental cell line, express pluripotency markers and have the ability to differentiate to the three germ layers. NOTCH1+/- iPSC can be used as a tool to study the cellular and molecular mechanisms underlying NOTCH1-associated human CHD.

Alagille syndrome (ALGS) is a multisystem disease with high variability in clinical features. ALGS is predominantly caused by pathogenic variants in the Notch ligand JAG1. An iPSC line, NCHi011-A, was generated from a ALGS patient with complex cardiac phenotypes consisting of pulmonic valve and branch pulmonary artery stenosis. NCHi011-A is heterozygous for a single base duplication causing a frameshift in the JAG1 gene. This iPSC line demonstrates normal cellular morphology, expression of pluripotency markers, trilineage differentiation potential, and identity to the source patient. NCHi011-A provides a resource for modeling ALGS and investigating the role of Notch signaling in the disease.

Down syndrome is a genetic anomaly that manifests when there is a mistake during cell division, resulting in an additional chromosome 21. Down syndrome can impact cognitive capabilities and physical development, giving rise to diverse developmental disparities and an elevated likelihood of certain health issues. The iPSC line NCHi010-A was generated from peripheral blood mononuclear cells of a 6-year-old female with Down syndrome and without congenital heart disease using Sendai virus reprogramming. NCHi010-A displayed a morphology of pluripotent stem cells, expressed pluripotency markers, retained trisomy 21 karyotype, and demonstrated potential to differentiate into cells representative of the three germ layers.

Alagille syndrome (ALGS) is an autosomal dominant disease affecting the liver, heart and other organs with high variability. About 95% of ALGS cases are associated with pathogenic variants in JAG1, encoding the Jagged1 ligand that binds to Notch receptors. The iPSC line NCHi012-A was derived from an ALGS patient with cholestatic liver disease and mild pulmonary stenosis, who is heterozygous for a 2 bp deletion in the JAG1 coding sequence. We report here an initial characterization of NCHi012-A to evaluate its morphology, pluripotency, differentiation potential, genotype, karyotype and identity to the source patient.