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Biology subjects

Bellin, M.

Publications and source records attributed to Bellin, M..

7 recordsLinked to original sources

Industrialization of three-dimensional hiPSC-cardiac microtissues for high-throughput cardiac safety and drug discovery screening

Current cardiac cell models for drug screening often face a trade-off between cellular maturity and achieving high throughput. While three-dimensional human induced pluripotent stem cell-based heart models typically exhibit more adult-like features, their application is hindered by the need for large cell numbers or complex equipment. Here, we developed cost-effective methods to scale up production of three-dimensional cardiac microtissues (cMTs) containing three cardiac cell types, and assess calcium transients and action potential metrics for high-throughput screening (HTS). Automating the procedure revealed reproducible drug responsiveness and predictive accuracy in a reference compound screen. Furthermore, an arrhythmic phenotype was reliably triggered in cMTs containing cardiomyocytes with a RYR2 mutation. A screen of FDA-approved drugs identified 17 drugs that rescued the arrhythmic phenotype. Our findings underscore the scalability of cMTs and their utility in disease modelling and HTS. The advanced "technology-readiness-level" of cMTs supports their regulatory uptake and acceptance within the pharmaceutical industry.

cell biology↗

Identification of molecular and functional subtypes using chronic pancreatitis patient-derived organoid models

Chronic pancreatitis (CP) affects [~]3 million people worldwide, yet altering the course of disease is challenging. We developed a patient-derived organoid (PDO) platform to investigate the molecular pathogenesis of this disease and identify therapeutic strategies. We generated 36 PDOs from patients with idiopathic, hereditary, and alcohol-related CP with high genetic concordance. PDOs retained inflammation-associated transcriptional and proteomic features. Transcriptomic profiling revealed three molecular subtypes of CP independent of etiology. We discovered widespread dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in half of the CP PDOs, including those with wildtype CFTR. Clinically available CFTR modulators stabilized mutant or wildtype CFTR, restored CFTR function, and decreased mitogenic and inflammatory signaling. This work provides the first comprehensive PDO platform for modeling CP. We demonstrate the utility of this platform for precision therapeutic investigations. Our findings reveal CFTR modulators as a broadly applicable and effective therapeutic strategy.

cell biology↗

Gain of chromosome region 1q31.3 in human iPSCs confers growth advantage and alters contraction in derivative cardiomyocytes

hPSCs can acquire chromosomal aberrations such as copy number variations during prolonged maintenance in vitro, conferring growth advantages. However, the effect of these culture-acquired mutations on the phenotypes of the differentiated hPSCs is largely unstudied. Here, we identified mosaicism in a hPSC line in which some cells showed a gain of chromosome 1q31.3. We subcloned the wild-type and variant hPSCs and could maintain both as stable lines. While both variant and wildtype lines differentiated efficiently to cardiomyocytes (hPSC-CMs), molecular analysis revealed the variant hPSC-CMs had increased expression of TNNT2, a gene encoding one of the major sarcomere proteins mediating cardiomyocyte contractility and located within the gained chromosome 1q region. Moreover, the variant hPSC-CMs showed altered contraction kinetics. Together these results highlight the importance of careful monitoring of chromosome aberrations in hPSC lines as these could have confounding effects in various applications such as disease modelling and drug discovery.

cell biology↗

Venom protection by antibody from a snakebite hyperimmune subject

Snake envenomation is a neglected tropical disease, causing >100,000 deaths and 300,000 permanent disabilities in humans annually. Could monoclonal antibody technology provide a solution? Here, we recover Centi-3FTX-D09, a potent broadly neutralizing antivenom antibody from the B-cell memory of a human subject with snake venom exposure. Centi-3FTX-D09 recognized a conserved neutralizing epitope on long 3-finger toxins (3FTXs), a dominant snake neurotoxin. Crystal structures of Centi-3FTX-D09 in complex with 3FTXs from mamba, taipan, krait, and cobra revealed epitope mimicry of the interface between these neurotoxins and their host target, the nicotinic acetylcholine receptor. Centi-3FTX-D09 provided in-vivo protection against diverse recombinant long 3FTXs, in-vivo rescue from whole venom challenge from cobras, black mamba, and king cobra, and, when combined with the phospholipase inhibitor varespladib, in-vivo protection extending to a majority of tested elapid venoms. Thus, a single antibody can broadly neutralize long neurotoxins and contribute to broad protection from envenomation.

immunology↗

Elevated islet prohormone ratios as indicators of insulin dependency in islet transplant recipients

Autologous pancreatic islet transplantation is an established therapy for patients with chronic pancreatitis. However, the long-term transplant outcomes are modest. Identifying indicators of graft function will aid the preservation of transplanted islets and glycemic control. To this end, we analyzed beta cell prohormone peptide levels in a retrospective cohort of total pancreatectomy autologous islet transplant patients (n=28). Proinsulin-to-C-peptide (PI/C) and proIAPP-to-total IAPP (proIAPP/IAPP) ratios measured at 3 months post-transplant were significantly higher in patients who remained insulin dependent at 1 year follow-up. In a mouse model of human islet transplantation, recipient mice that later became hyperglycemic displayed significantly higher PI/C ratios than mice that remained normoglycemic. Histological analysis of islet grafts showed reduced insulin- and proinsulin-positive area, but elevated glucagon-positive area in grafts that experienced greater secretory demand. Increased prohormone convertase 1/3 was detected in glucagon-positive cells, and glucagon-like peptide 1 (GLP-1) area was elevated in grafts from mice that displayed hyperglycemia or elevated plasma PI/C ratios, demonstrating intra-islet incretin production in metabolically challenged human islet grafts. These data indicate that in failing grafts, alpha cell prohormone processing is likely altered, and incomplete beta cell prohormone processing may be an early indicator of insulin dependency.

pathology↗

Renalase is a novel tissue and serological biomarker in pancreatic ductal adenocarcinoma

Dysregulated expression of the secretory protein renalase (RNLS) can promote pancreatic ductal adenocarcinoma (PDAC) growth in animal models. We characterized RNLS expression in premalignant and malignant PDAC tissue and investigated whether plasma RNLS levels corresponded to clinical PDAC characteristics. RNLS immunohistochemistry was used to determine the presence and distribution of RNLS in normal pancreas, chronic pancreatitis, PDAC precursor lesions, and PDAC tissues. Associations between pretreatment plasma RNLS and PDAC clinical status were assessed in patients with varied clinical stages of PDAC and included tumor characteristics, surgical resection in locally advanced/borderline resectable PDAC, and overall survival. Data were retrospectively obtained and correlated using non-parametric analysis. Mild to no RNLS was detected by histochemistry in the normal pancreas in the absence of abdominal trauma. In chronic pancreatitis, RNLS immunoreactivity localized to peri-acinar spindle-shaped cells in some samples. It was also widely present in PDAC precursor lesions and PDAC tissue. Among 240 patients with PDAC, elevated plasma RNLS levels were associated with worse tumor characteristics, including greater angiolymphatic invasion (80.0% vs. 58.1%, p = 0.012) and greater node positive disease (76.5% vs. 56.5%, p = 0.024). Overall survival was worse in patients with high plasma RNLS levels with median follow-up of 27.70 months vs. 65.03 months (p < 0.001). RNLS levels also predicted whether patients with locally advanced/borderline resectable (LA/BR) PDAC underwent resection (AUC 0.674; 95%CI 0.42-0.82, p = 0.04). Overall tissue RNLS was increased in both premalignant and malignant PDAC tissues compared to normal pancreas. Elevated plasma RNLS levels were associated with advanced tumor characteristics, decreased overall survival, and reduced resectability in patients with LA/BR PDAC. These studies show that RNLS levels are increased in premalignant pancreatic tissues and that its levels in plasma correspond to the clinical behavior of PDAC.

cancer biology↗

Versatile open software to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo

Contraction of muscle reflects its physiological state. Methods to quantify contraction are often complex, expensive and tailored to specific models or recording conditions, or require specialist knowledge for data extraction. Here we describe an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. MUSCLEMOTION allowed rapid and easy measurement of contractility in (i) single cardiomyocytes from primary adult heart and human pluripotent stem cells, (ii) multicellular 2D-cardiomyocyte cultures, 3D engineered heart tissues and cardiac organoids/microtissues in vitro and (iii) intact hearts of zebrafish and humans in vivo. Good correlation was found with conventional measures of contraction in each system. Thus, using a single method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell- and animal models and human echocardiograms.

physiology↗