Combined GWAS and single cell transcriptomics uncover the underlying genes and cell types in disorders of gut-brain interaction.

Disorders of gut-brain interaction (DGBIs), formerly known as functional gastrointestinal disorders, are extremely common and historically difficult to manage. This is largely because their cellular and molecular mechanisms have remained poorly understood and understudied. One approach to unravel the molecular underpinnings of complex disorders such as DGBIs is performing genome wide association studies (GWASs). However, due to the heterogenous and non-specific nature of GI symptoms, it has been difficult to accurately classify cases and controls. Thus, to perform reliable studies, we need to access large patient populations which has been difficult to date. Here, we leveraged the UK Biobank (UKBB) database, containing genetic and medical record data of over half a million individuals, to perform GWAS for five DGBI categories: functional chest pain, functional diarrhea, functional dyspepsia, functional dysphagia, and functional fecal incontinence. By applying strict inclusion and exclusion criteria, we resolved patient populations and identified genes significantly associated with each condition. Leveraging multiple human single-cell RNA-sequencing datasets, we found that the disease associated genes were highly expressed in enteric neurons, which innervate and control GI functions. Further expression and association testing-based analyses revealed specific enteric neuron subtypes consistently linked with each DGBI. Furthermore, protein-protein interaction analysis of each of the disease associated genes revealed protein networks specific to each DGBI, including hedgehog signaling for functional chest pain and neuronal function and neurotransmission for functional diarrhea and functional dyspepsia. Finally, through retrospective medical record analysis we found that drugs that inhibit these networks are associated with an increased disease risk, including serine/threonine kinase 32B drugs for functional chest pain, solute carrier organic anion transporter family member 4C1, mitogen-activated protein kinase 6, and dual serine/threonine and tyrosine protein kinase drugs for functional dyspepsia, and serotonin transporter drugs for functional diarrhea. This study presents a robust strategy for uncovering the tissues, cell types, and genes involved in DGBIs, presenting novel predictions of the mechanisms underlying these historically intractable and poorly understood diseases.

Deriving Schwann cells from hPSCs enables disease modeling and drug discovery for diabetic peripheral neuropathy.

Schwann cells (SCs) are the primary glia of the peripheral nervous system. SCs are involved in many debilitating disorders, including diabetic peripheral neuropathy (DPN). Here, we present a strategy for deriving SCs from human pluripotent stem cells (hPSCs) that enables comprehensive studies of SC development, physiology, and disease. hPSC-derived SCs recapitulate the molecular features of primary SCs and are capable of in vitro and in vivo myelination. We established a model of DPN that revealed the selective vulnerability of SCs to high glucose. We performed a high-throughput screen and found that an antidepressant drug, bupropion, counteracts glucotoxicity in SCs. Treatment of hyperglycemic mice with bupropion prevents their sensory dysfunction, SC death, and myelin damage. Further, our retrospective analysis of health records revealed that bupropion treatment is associated with a lower incidence of neuropathy among diabetic patients. These results highlight the power of this approach for identifying therapeutic candidates for DPN.

Tyrosine kinase inhibitor-associated ventricular arrhythmias: a case series and review of literature.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) have been increasingly used as first-line therapy in hematologic and solid-organ malignancies. Multiple TKIs have been linked with the development of cardiovascular complications, especially atrial arrhythmias, but data on ventricular arrhythmias (VAs) is scarce. METHODS: Herein we describe five detailed cases of VAs related to TKI use in patients with varied baseline cardiovascular risk factors between 2019 and 2022 at three centers. Individual chart review was conducted retrospectively. RESULTS: Patient ages ranged from 43 to 83 years. Three patients were on Bruton's TKI (2 ibrutinib and 1 zanubrutinib) at the time of VAs; other TKIs involved were afatinib and dasatinib. Three patients had a high burden of non-sustained ventricular tachycardia (NSVT) requiring interventions, whereas two patients had sustained VAs. While all patients in our case series had significant improvement in VA burden after TKI cessation, two patients required new long-term antiarrhythmic drug therapy, and one had an implantable defibrillator cardioverter (ICD) placed due to persistent VAs after cessation of TKI therapy. One patient reinitiated TKI therapy after control of arrhythmia was achieved with antiarrhythmic drug therapy. CONCLUSIONS: Given the expanding long-term use of TKIs among a growing population of cancer patients, it is critical to acknowledge the association of TKIs with cardiovascular complications such as VAs, to characterize those at risk, and deploy preventive and therapeutic measures to avoid such complications and interference with oncologic therapy. Further efforts are warranted to develop monitoring protocols and optimal treatment strategies for TKI-induced VAs.

A dual SHOX2:GFP; MYH6:mCherry knockin hESC reporter line for derivation of human SAN-like cells.

The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and limitations in robust in vitro derivation methods. We developed a dual SHOX2:GFP; MYH6:mCherry knockin human embryonic stem cell (hESC) reporter line, which allows the identification and purification of SAN-like cells. Using this line, we performed several rounds of chemical screens and developed an efficient strategy to generate and purify hESC-derived SAN-like cells (hESC-SAN). The derived hESC-SAN cells display molecular and electrophysiological characteristics of bona fide nodal cells, which allowed exploration of their transcriptional profile at single-cell level. In sum, our dual reporter system facilitated an effective strategy for deriving human SAN-like cells, which can potentially be used for future disease modeling and drug discovery.

Relation of Cardiovascular Risk Factors to Mortality and Cardiovascular Events in Hospitalized Patients With Coronavirus Disease 2019 (from the Yale COVID-19 Cardiovascular Registry).

Individuals with established cardiovascular disease or a high burden of cardiovascular risk factors may be particularly vulnerable to develop complications from coronavirus disease 2019 (COVID-19). We conducted a prospective cohort study at a tertiary care center to identify risk factors for in-hospital mortality and major adverse cardiovascular events (MACE; a composite of myocardial infarction, stroke, new acute decompensated heart failure, venous thromboembolism, ventricular or atrial arrhythmia, pericardial effusion, or aborted cardiac arrest) among consecutively hospitalized adults with COVID-19, using multivariable binary logistic regression analysis. The study population comprised 586 COVID-19 positive patients. Median age was 67 (IQR: 55 to 80) years, 47.4% were female, and 36.7% had cardiovascular disease. Considering risk factors, 60.2% had hypertension, 39.8% diabetes, and 38.6% hyperlipidemia. Eighty-two individuals (14.0%) died in-hospital, and 135 (23.0%) experienced MACE. In a model adjusted for demographic characteristics, clinical presentation, and laboratory findings, age (odds ratio [OR], 1.28 per 5 years; 95% confidence interval [CI], 1.13 to 1.45), previous ventricular arrhythmia (OR, 18.97; 95% CI, 3.68 to 97.88), use of P2Y12-inhibitors (OR, 7.91; 95% CI, 1.64 to 38.17), higher C-reactive protein (OR, 1.81: 95% CI, 1.18 to 2.78), lower albumin (OR, 0.64: 95% CI, 0.47 to 0.86), and higher troponin T (OR, 1.84; 95% CI, 1.39 to 2.46) were associated with mortality (p <0.05). After adjustment for demographics, presentation, and laboratory findings, predictors of MACE were higher respiratory rates, altered mental status, and laboratory abnormalities, including higher troponin T (p <0.05). In conclusion, poor prognostic markers among hospitalized patients with COVID-19 included older age, pre-existing cardiovascular disease, respiratory failure, altered mental status, and higher troponin T concentrations.

Androgen Signaling Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men.

SARS-CoV-2 infection has led to a global health crisis, and yet our understanding of the disease and potential treatment options remains limited. The infection occurs through binding of the virus with angiotensin converting enzyme 2 (ACE2) on the cell membrane. Here, we established a screening strategy to identify drugs that reduce ACE2 levels in human embryonic stem cell (hESC)-derived cardiac cells and lung organoids. Target analysis of hit compounds revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression and protected hESC-derived lung organoids against SARS-CoV-2 infection. Finally, clinical data on COVID-19 patients demonstrated that prostate diseases, which are linked to elevated androgen, are significant risk factors and that genetic variants that increase androgen levels are associated with higher disease severity. These findings offer insights on the mechanism of disproportionate disease susceptibility in men and identify antiandrogenic drugs as candidate therapeutics for COVID-19.

Androgen Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a global health crisis, and yet our understanding of the disease pathophysiology and potential treatment options remains limited. SARS-CoV-2 infection occurs through binding and internalization of the viral spike protein to angiotensin converting enzyme 2 (ACE2) on the host cell membrane. Lethal complications are caused by damage and failure of vital organs that express high levels of ACE2, including the lungs, the heart and the kidneys. Here, we established a high-throughput drug screening strategy to identify therapeutic candidates that reduce ACE2 levels in human embryonic stem cell (hESC) derived cardiac cells. Drug target analysis of validated hit compounds, including 5 alpha reductase inhibitors, revealed androgen signaling as a key modulator of ACE2 levels. Treatment with the 5 alpha reductase inhibitor dutasteride reduced ACE2 levels and internalization of recombinant spike receptor binding domain (Spike-RBD) in hESC-derived cardiac cells and human alveolar epithelial cells. Finally, clinical data on coronavirus disease 2019 (COVID-19) patients demonstrated that abnormal androgen states are significantly associated with severe disease complications and cardiac injury as measured by blood troponin T levels. These findings provide important insights on the mechanism of increased disease susceptibility in male COVID-19 patients and identify androgen receptor inhibition as a potential therapeutic strategy.

A human embryonic stem cell reporter line for monitoring chemical-induced cardiotoxicity.

AIMS: Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs. METHODS AND RESULTS: We used CRISPR/Cas9 to knock a mCherry reporter gene into the MYH6 locus of hESC lines, H1 and H9, enabling real-time monitoring of the generation of CMs. MYH6:mCherry+ cells express atrial or ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20 days of differentiation, MYH6:mCherry+ cells show features characteristic of human CMs and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiac arrhythmia. CONCLUSION: We created two MYH6:mCherry hESC reporter lines and documented the application of these lines for disease modelling relevant to cardiomyocyte biology.

Metastable Atrial State Underlies the Primary Genetic Substrate for MYL4 Mutation-Associated Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. METHODS: Using an MYH6:mCherry knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell-derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human MYL4 mutants, one of the few definitive genetic causes of AF. To explore non-cell-autonomous components of AF substrate, we also created a zebrafish Myl4 knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. RESULTS: There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)-related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in MYL4 mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. CONCLUSIONS: These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.

Cardioprotective effects of omega-3 fatty acids and ascorbic acid improve regenerative capacity of embryonic stem cell-derived cardiac lineage cells.

One of the major issues in cell therapy of myocardial infarction (MI) is early death of engrafted cells in a harsh oxidative stress environment, which limits the potential therapeutic utility of this strategy in the clinical setting. Increasing evidence implicates beneficial effects of omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and ascorbic acid (AA) in cardiovascular diseases, in particular their role in ameliorating fibrosis. In the current study, we aim to assess the cytoprotective role of EPA + DHA and AA in protecting embryonic stem cell (ESC)-derived cardiac lineage cells and amelioration of fibrosis. Herein, we have shown that preincubation of the cells with EPA + DHA + AA prior to H2 O2 treatment attenuated generation of reactive oxygen species (ROS) and enhanced cell viability. Gene expression analysis revealed that preincubation with EPA + DHA + AA followed by H2 O2 treatment, upregulated heme oxygenase-1 (HO-1) along with cardiac markers (GATA4, myosin heavy chain, α isoform [MYH6]), connexin 43 [CX43]) and attenuated oxidative stress-induced upregulation of fibroblast markers (vimentin and collagen type 1 [Col1]). Alterations in gene expression patterns were followed by marked elevation of cardiac troponin (TNNT2) positive cells and reduced numbers of vimentin positive cells. An injection of EPA + DHA + AA-pretreated ESC-derived cardiac lineage cells into the ischemic myocardium of a rat model of MI significantly reduced fibrosis compared to the vehicle group. This study provided evidence that EPA + DHA + AA may be an appropriate preincubation regimen for regenerative purposes. © 2019 BioFactors, 45(3):427-438, 2019.

A hPSC-based platform to discover gene-environment interactions that impact human ß-cell and dopamine neuron survival.

Common disorders, including diabetes and Parkinson's disease, are caused by a combination of environmental factors and genetic susceptibility. However, defining the mechanisms underlying gene-environment interactions has been challenging due to the lack of a suitable experimental platform. Using pancreatic ß-like cells derived from human pluripotent stem cells (hPSCs), we discovered that a commonly used pesticide, propargite, induces pancreatic ß-cell death, a pathological hallmark of diabetes. Screening a panel of diverse hPSC-derived cell types we extended this observation to a similar susceptibility in midbrain dopamine neurons, a cell type affected in Parkinson's disease. We assessed gene-environment interactions using isogenic hPSC lines for genetic variants associated with diabetes and Parkinson's disease. We found GSTT1-/- pancreatic ß-like cells and dopamine neurons were both hypersensitive to propargite-induced cell death. Our study identifies an environmental chemical that contributes to human ß-cell and dopamine neuron loss and validates a novel hPSC-based platform for determining gene-environment interactions.

Discovery of a drug candidate for GLIS3-associated diabetes.

GLIS3 mutations are associated with type 1, type 2, and neonatal diabetes, reflecting a key function for this gene in pancreatic ß-cell biology. Previous attempts to recapitulate disease-relevant phenotypes in GLIS3-/- ß-like cells have been unsuccessful. Here, we develop a "minimal component" protocol to generate late-stage pancreatic progenitors (PP2) that differentiate to mono-hormonal glucose-responding ß-like (PP2-ß) cells. Using this differentiation platform, we discover that GLIS3-/- hESCs show impaired differentiation, with significant death of PP2 and PP2-ß cells, without impacting the total endocrine pool. Furthermore, we perform a high-content chemical screen and identify a drug candidate that rescues mutant GLIS3-associated ß-cell death both in vitro and in vivo. Finally, we discovered that loss of GLIS3 causes ß-cell death, by activating the TGFß pathway. This study establishes an optimized directed differentiation protocol for modeling human ß-cell disease and identifies a drug candidate for treating a broad range of GLIS3-associated diabetic patients.

Prospective Isolation of ISL1+ Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy.

The LIM-homeodomain transcription factor ISL1 marks multipotent cardiac progenitors that give rise to cardiac muscle, endothelium, and smooth muscle cells. ISL1+ progenitors can be derived from human pluripotent stem cells, but the inability to efficiently isolate pure populations has limited their characterization. Using a genetic selection strategy, we were able to highly enrich ISL1+ cells derived from human embryonic stem cells. Comparative quantitative proteomic analysis of enriched ISL1+ cells identified ALCAM (CD166) as a surface marker that enabled the isolation of ISL1+ progenitor cells. ALCAM+/ISL1+ progenitors are multipotent and differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. Transplantation of ALCAM+ progenitors enhances tissue recovery, restores cardiac function, and improves angiogenesis through activation of AKT-MAPK signaling in a rat model of myocardial infarction, based on cardiac MRI and histology. Our study establishes an efficient method for scalable purification of human ISL1+ cardiac precursor cells for therapeutic applications.

ROCKII inhibition promotes the maturation of human pancreatic beta-like cells.

Diabetes is linked to loss of pancreatic beta-cells. Pluripotent stem cells offer a valuable source of human beta-cells for basic studies of their biology and translational applications. However, the signalling pathways that regulate beta-cell development and functional maturation are not fully understood. Here we report a high content chemical screen, revealing that H1152, a ROCK inhibitor, promotes the robust generation of insulin-expressing cells from multiple hPSC lines. The insulin expressing cells obtained after H1152 treatment show increased expression of mature beta cell markers and improved glucose stimulated insulin secretion. Moreover, the H1152-treated beta-like cells show enhanced glucose stimulated insulin secretion and increased capacity to maintain glucose homeostasis after transplantation. Conditional gene knockdown reveals that inhibition of ROCKII promotes the generation and maturation of glucose-responding cells. This study provides a strategy to promote human beta-cell maturation and identifies an unexpected role for the ROCKII pathway in the development and maturation of beta-like cells.Our incomplete understanding of how pancreatic beta cells form limits the generation of beta-like cells from human pluripotent stem cells (hPSC). Here, the authors identify a ROCKII inhibitor H1152 as increasing insulin secreting cells from hPSCs and improving beta-cell maturation on transplantation in vivo.

Transient Activation of Reprogramming Transcription Factors Using Protein Transduction Facilitates Conversion of Human Fibroblasts Toward Cardiomyocyte-Like Cells.

Derivation of cardiomyocytes directly from patients' own fibroblasts could offer a new therapeutic approach for those with ischemic heart disease. An essential step toward clinical application is to establish safe conversion of human fibroblasts into a cardiac fate. Here we aimed to efficiently and safely generate cardiomyocytes from human fibroblasts by direct delivery of reprogramming recombinant cell permeant form of reprogramming proteins followed by cardio-inductive signals. Human fetal and adult fibroblasts were transiently exposed to transactivator of transcription-fused recombinant OCT4, SOX2, KLF4 and c-MYC for 2 weeks and then were directly differentiated toward protein-induced cardiomyocyte-like cells (p-iCLCs) in a cardiac fate niche, carried out by treatment with a set of cardiogenic small molecules (sequential treatment of Chir, and IWP-2, SB431542 and purmorphamine). The cells showed cardiac phenotype over a period of 3 weeks without first undergoing reprogramming into or through a pluripotent intermediate, shown by lack of expression of key pluripotency markers. p-iCLCs exhibited cardiac features at both the gene and protein levels. Our study provides an alternative method for the generation of p-iCLCs which shortcut reprogramming toward allogeneic cardiomyocytes in a safe and efficient manner and could facilitate generation of genetic material-free cardiomyocytes.

Isolation and characterization of cardiogenic, stem-like cardiac precursors from heart samples of patients with congenital heart disease.

AIMS: Regenerative therapies based on resident human cardiac progenitor cells (hCPCs) are a promising alternative to medical treatments for patients with myocardial infarction. However, hCPCs are rare in human heart and finding efficient source and proper surface marker for isolation of these cells would make them a good candidate for therapy. MAIN METHODS: We have isolated 5.34∗10(6)±2.04∗10(5)/g viable cells from 35 heart tissue samples of 23 patients with congenital heart disease obtained during their heart surgery along with 6 samples from 3 normal subjects during cardiac biopsy. KEY FINDINGS: According to FACS analysis, younger ages, atrial specimen and disease with increased pulmonary vascular resistance were associated with higher percentage of c-kit(+) (CD117) hCPCs. Analysis for other stemness markers revealed increased CD133(+) cells in the hearts of patients with congenital heart disease. By using both immune-labeling and PCR, we demonstrated that these cells express key cardiac lineage and endothelial transcription factors and structural proteins during in vitro differentiation and do express stemness transcription factors in undifferentiated state. Another novel datum of potentially relevant interest is their ability in promoting greater myocardial regeneration and better survival in rat model of myocardial infarction following transplantation. SIGNIFICANCE: Our results could provide evidence for conditions associated with enriched hCPCs in patients with congenital heart disease. Moreover, we showed presence of a significant number of CD133 expressing cardiogenic stem-like cardiac precursors in the heart of patients with congenital heart disease, which could be isolated and stored for future regenerative therapies in these patients.

Cellular and molecular characterization of human cardiac stem cells reveals key features essential for their function and safety.

Cell therapy of heart diseases is emerging as one of the most promising known treatments in recent years. Transplantation of cardiac stem cells (CSCs) may be one of the best strategies to cure adult or pediatric heart diseases. As these patient-derived stem cells need to be isolated from small heart biopsies, it is important to select the best isolation method and CSC subpopulation with the best cardiogenic functionality. We employed three different protocols including c-KIT(+) cell sorting, clonogenic expansion, and explants culture to isolate c-KIT(+) cells, clonogenic expansion-derived cells (CEDCs), and cardiosphere-derived cells (CDCs), respectively. Evaluation of isolated CSC characteristics in vitro and after rat myocardial infarction (MI) model transplantation revealed that although c-KIT(+) and CDCs had higher MI regenerative potential, CEDCs had more commitment into cardiomyocytes and needed lower passages that were essential to reach a definite cell count. Furthermore, genome-wide expression analysis showed that subsequent passages caused changes in characteristics of cells, downregulation of cell cycle-related genes, and upregulation of differentiation and carcinogenic genes, which might lead to senescence, commitment, and possible tumorigenicity of the cells. Because of different properties of CSC subpopulations, we suggest that appropriate CSCs subpopulation should be chosen based on their experimental or clinical use.