Biology subjects

Fernandes, I.

Publications and source records attributed to Fernandes, I..

11 recordsLinked to original sources

bioRxiv2025

Generation of Valvular Interstitial Cells from Human Pluripotent Stem Cells

Heart valves are living structures whose sophisticated functions are mediated by a specialized population of mesenchymal cells known as valvular interstitial cells (VICs). Given their central role in valve homeostasis, VICs represent a promising cell population for studying heart valve diseases and developing novel therapies to treat them. Here, we describe a strategy for generating VICs from human pluripotent stem cells (hPSCs) by stage-specific manipulation of developmental signalling pathways. Our results demonstrate that hPSC-derived VICs show a high transcriptional similarity to primary human fetal VICs and can secrete key proteins of the valve extracellular matrix. We further investigate the heterogeneity of hPSC-derived VICs and identify two major subpopulations with distinct molecular and functional properties, mirroring the cellular diversity observed in vivo. Finally, we utilize an in vitro model of Noonan syndrome to demonstrate that hPSC-derived VICs can accurately recapitulate key aspects of valve disease. Collectively, these findings provide a reproducible method for the scaled generation of bona fide hPSC-derived VICs and establish their utility in disease modelling and tissue engineering applications. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- We established a robust platform to generate bona fide valvular interstitial cells (VICs) from human pluripotent stem cells (hPSCs), recapitulating native VIC identity and function. - We delineated signaling pathways that promote the development of two distinct VIC subsets and identified a surface marker to distinguish between them. What are the clinical implications?- A renewable, human-specific source of VICs enables precise mechanistic studies of valve development and disease that are not possible with limited surgical specimens or animal models. - This platform creates opportunities for therapeutic applications, including drug discovery and tissue engineering approaches for valve repair.

cell biology↗

bioRxiv2025

Growth Factor-Based Manufacturing of Human Pluripotent Stem Cell-Derived Cardiomyocytes Using the Vertical Wheel Bioreactor System

BackgroundMultiple protocols have been reported for the large-scale generation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in bioreactors using small molecules; however, no comparable bioreactor-based methods have been established using growth factors. This is despite evidence that differentiation with optimized concentrations of BMP4, Activin A, and bFGF offers finer control of cardiomyocyte phenotype. Here, we develop scalable hPSC expansion and growth factor-based cardiac differentiation protocols using the vertical wheel bioreactor (VWBR) system. Methods and ResultsThe expansion of undifferentiated hPSCs was first optimized in 100 mL VWBRs by varying parameters, including starting cell seeding density, agitation rate, and media exchange schedule. Proliferation, viability, aggregate diameter, media metabolites, and pluripotency were assessed during hPSC expansion. Next, we evaluated the effects of undifferentiated hPSC culture conditions on subsequent cardiomyocyte differentiation potential. We found that hPSCs expanded in static culture or in VWBRs at different densities and agitation rates all differentiated into hPSC-CM populations of similar cardiac purity; however, cardiomyocyte yields were initially lower when VWBR-expanded hPSCs were used. We compared the differentiation kinetics of hPSCs expanded in VWBRs to conventional 2D culture and found that the former had accelerated mesodermal commitment and significantly greater cKit+/CXCR4+/PDGRF- cell formation during differentiation. Modifying our aggregation and mesoderm induction steps improved cell yields and enabled reliable production of >1x106 cells/mL cardiac troponin T+ (cTnT) hPSC-CMs. Highlighting the versatility of our growth factor-based system, variation in the BMP4:Activin A ratio enabled a second heart field-like differentiation and generation of atrial-like cardiomyocytes in VWBRs. We further show that our expansion and differentiation protocols are reproducible and economical in 500 mL VWBRs, yielding on average 1.11x106 hPSC-CMs/mL at a mean purity of 93% cTnT+. Characterization of VWBR produced hPSC-CM force generation, action potentials, and intercellular calcium transients confirmed the expected phenotype of ventricular-like cells. Lastly, VWBR produced hPSC-CMs robustly engrafted in the infarcted guinea pig myocardium, supporting use as a cell therapy product. ConclusionsThis novel bioreactor-based protocol will enable cardiac cell therapy and tissue engineering applications by providing scalable and consistent production of hPSC-derived cardiac cell products.

cell biology↗

bioRxiv2025

Epicardial extracellular vesicles modulate gene expression following ischemia-reperfusion injury in heart-on-a-chip

The epicardium is an essential regulator of cardiac development, homeostasis, and injury, yet the composition and impacts of epicardial cell-secreted extracellular vesicles (EVs) remain incompletely understood. Here, we harness an epicardial Biowire platform integrating human stem cell derived epicardial cells with functional myocardium and apply transcriptomics to reveal enriched EV transport in tissues containing epicardial cells. Profiling epicardial-EVs identified key miRNAs and their conservation through stimulated epithelial-to-mesenchymal transition. Supplementation of epicardial-EVs to tissues undergoing ischemia-reperfusion injury influenced gene expression associated with reduction of extracellular matrix remodeling, fibroblast activation, and suppression of cell-ECM interactions. Correlation of EV-miRNAs with mRNA targets highlighted the role of miR-30d-5p, miR-9-5p, miR-16-5p, and the let-7 family in moderating deleterious fibrotic activation and matrix remodelling during myocardial injury in vitro. TeaserCells from the hearts outer surface secrete tiny packages of biomolecules that influence how the heart responds to injury

bioengineering↗

bioRxiv2025

Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

Scalable generation of functionally mature hepatocytes from human pluripotent stem cells (hPSCs) is vital for regenerative medicine, disease modeling, and drug screening. We present a modular differentiation platform that enables serial expansion and cryopreservation of hepatic progenitors (hepatoblasts; HBs), supporting efficient downstream maturation. By modulating bile acid signaling with FGF19 and culturing under hypoxia, we preserved HB proliferation and bipotency. Subsequent thyroid hormone treatment induced alpha-fetoprotein-negative, albumin-positive hepatocytes, while WNT pathway modulation promoted zonal identity, mimicking in vivo hepatic metabolic heterogeneity. Zonation was confirmed by differential drug-metabolizing enzyme activity, mitochondrial function, and enhanced engraftment in a liver injury mouse model. To evaluate translational relevance, mature hepatocytes were incorporated into extrusion-based 3D bioprinted constructs, which maintained hepatic viability and function. This integrated strategy of combining progenitor cell banking, zonation control, and tissue engineering offers a scalable and clinically relevant approach for generating functional human liver tissue suitable for therapeutic development.

cell biology↗

bioRxiv2024

Effect of high-intensity anaerobic exercise on electrocortical activity in athletes and non-athletes

AimThe present study aims to verify the information processing in athletes through electroencephalography, analyze cortical areas responsible for cognitive functions related to attentional processing of visual stimuli, and investigate motor activitys influence on cognitive aspects. Material and MethodsThe sample consisted of 29 subjects, divided into an experimental group (n = 13 modern pentathlon athletes) and a control group (n = 16 non-athletes). We collected the electrocortical activity before and after the Wingate Anaerobic Test. During the electrophysiological measures, the volunteers performed a saccadic eye movement paradigm. They also performed cognitive tasks, resting heart rate, and anthropometric measurements. ResultsA mixed ANOVA was applied to analyze the statistical differences between groups (athletes and control) and moments (before and after exercise) for F3, F4, P3, and P4 electrodes during rest one and task (pre-stimulus GO). There was an interaction for the group vs. moment factors in F3 [F = 17,129; p = 0,000; {eta}{superscript 2} = 0.512], F4 [F = 22,774; p = 0,000; {eta}{superscript 2} = 0.510], P3 [F = 11,429; p = 0,001; {eta}{superscript 2} = 0.405], and P4 electrodes [F = 18,651; p = 0,000; {eta}{superscript 2} = 0.379]. We found the main effect for group factors in the frontal and parietal electrodes of the right hemisphere (F4 and P4) and a main effect of the moment factor on the frontal (F3 and F4) and parietal (P3 and P4) electrodes. There was an interaction between the group vs. moment factors for the reaction time. The groups were different in Peak Power (Watts/kg), Average Power (Watts/kg), Fatigue Index (%), and Maximum Power (ms). ConclusionsWe identified chronic effects of exercise training on the cortical activity of modern pentathlon athletes, read-through differences in absolute alpha power, and acute effects of a high-intensity exercise session for athletes and non-athletes for electrocortical and behavioral responses.

neuroscience↗

bioRxiv2024

Attention and working memory investigated through the P300 component in children practicing Karate at different stages of biological maturation

AimTo investigate attention and working memory, comparing children practice Karate and non-Karate practitioners at different stages of biological maturation through the amplitude and latency of the P300 component during the execution of a Go/No-Go paradigm. Material and MethodsThe P300 was analyzed for Fz, Cz, and Pz electrodes in 80 participants separated in two groups: an Karate practitioners group comprising Karate practitioners and comprising non-Karate practitioners. Each group was further subdivided according to the biological maturation range defined by Peak Height Velocity. In addition, the participants performed a Go/No-Go paradigm to measure amplitude and latency. ResultsThe EEG analysis showed Ffr electrodes Pz and Cz, an interaction was found between group and Peak Height Velocity for the amplitude variable (respectively: F = 45.858; d = 0.38; p < 0.001 / F = 10.411; d = 0.17; p = 0.004). For the Fz electrode, a main effect was found between group and Peak Height Velocity (respectively: F = 40.330; d = 0.34; p = 0.010 / F = 36.730; d = 0.30; p = 0.012) for the variable amplitude and latency. main effect between group and Peak Height Velocity (respectively: F = 7.719; d = 0.14; p = 0.012 / F = 38.370; d = 0.31; p = 0.010). ConclusionsIn general, it is possible to conclude that participants in the Karate practitioners group exhibited electrocortical measures corresponding to greater efficiency in decision-making and attention processes, motor planning, working memory, attention allocation, motor execution, and greater attentional engagement. It was also demonstrated that, despite the children being at very close chronological ages, their biological maturation differed.

neuroscience↗

bioRxiv2023

Modeling cardiac fibroblast heterogeneity from human pluripotent stem cell-derived epicardial cells

Abstract (Summary) Cardiac fibroblasts play an essential role in the development of the heart and have been implicated in disease progression in the context of fibrosis and regeneration. Here, we established a simple organoid culture platform using human pluripotent stem cell-derived epicardial cells and ventricular cardiomyocytes to study cardiac fibroblasts development, maturation and heterogeneity under normal conditions and following treatment with pathological stimuli. We demonstrated that this system models the early interactions between epicardial cells and cardiomyocytes to generate a population of fibroblasts that recapitulates many aspects of fibroblast behaviour in vivo including changes associated with maturation and in response to pathological stimuli associated with cardiac injury. Using single cell transcriptomics, we show that the hPSC-derived organoid fibroblast population displays a high degree of heterogeneity that approximates the heterogeneity of populations in both the normal and diseased human heart. Additionally, we identified a unique subpopulation of fibroblasts possessing reparative features previously characterized in the hearts of model organisms. Taken together, our system recapitulates many aspects of human cardiac fibroblast specification, development and maturation providing a platform to investigate the role of these cells in human cardiovascular development and disease.

cell biology↗

bioRxiv2023

Decrease in reaction time for volleyball athletes during saccadic eye movement task: An associative analysis with evoked potentials

AimThe current study investigated the differences to event-related potential and reaction time under two groups (athletes vs. non-athletes). Material and MethodsThe P300 was analyzed for Fz, Cz and Pz electrodes in thirty-one healthy volunteers divided into two groups (volleyball athletes and non-athletes). In addition, the participants performed a saccadic eye movement task to measure reaction time. ResultsThe EEG analysis showed that the athletes in comparison to the no-athletes has differences of the P300 in the frontal area (p=0.021). In relation to reaction time, the results show lower reaction time for athletes (p=0.001). ConclusionsThe volleyball athletes may present a greater allocation of attention during the execution of the inhibition task, since they have a lower reaction time for responses when compared to non-athletes.

neuroscience↗

bioRxiv2022

Non-invasive quantification of contractile dynamics in cardiac cells, spheroids and organs-on-a-chip using high frequency ultrasound

Cell-based models that mimic in vivo heart physiology are poised to make significant advances in cardiac disease modeling and drug discovery. In these systems, cardiomyocyte (CM) contractility is an important functional metric, but current measurement methods are inaccurate, low-throughput, or require complex set-ups. To address this need, we developed a standalone non-invasive, label-free ultrasound technique operating at 40-200 MHz to measure the beat rate, beat rhythm, and force of contraction of cardiac models, ranging from single adult CMs to 3D microtissue constructs in standard cell culture formats. The high temporal resolution of 1000 fps resolved the beat profile of single mouse CMs paced at up to 9 Hz, revealing limitations of lower speed optical based measurements to resolve beat kinetics or characterize aberrant beats. Coupling of ultrasound with traction force microscopy enabled the measurement of CM longitudinal modulus and facile estimation of adult mouse CM contractile forces of 2.34 {+/-} 1.40 N, comparable to more complex measurement techniques. Similarly, measurements of beat rate, rhythm, and drug responses of CM spheroid and microtissue models were demonstrated. In conclusion, ultrasound can be used for the rapid characterization of CM contractile function in a wide range of commonly-studied configurations ranging from single cells to 3D tissue constructs using standard well plates, with applications in cardiac drug discovery and cardiotoxicity evaluation.

bioengineering↗

bioRxiv2022

Data-driven machine learning for pattern recognition supports environmental quality prediction for irrigated rice in Brazil

The sustainability of irrigated rice (Oryza sativa L.) production systems in Brazilian tropical region highly depends on the success of developing stable cultivars. To achieve this goal, many steps in product development must address the environmental variability and genotype by environment interactions (GE), which makes difficult the design and development of local-specific adapted cultivars. Thus, the adoption of new strategies for characterizing environmental-phenotype relations are the key for optimizing this process. In addition, it could also benefit post-breeding stages of seed production. To overcome this situation, we implemented a data-driven approach to link environmental characterization to yield clustering using historical data (1982-2017, 31 locations, 471 genotypes), 42 envirotyping covariables and machine learning (ML), combining two unsupervised (K-means and decision tree models, DTC) algorithms. Additionally, linear mixed models (LMM) were applied to explore the relations between the outcomes of our approach and GE analysis for irrigated rice yield in Brazilian tropical region. Four environments were identified: Very Low Yield (1.7 Mg.ha-1), Low Yield (5.1 Mg.ha-1), High Yield (7.2 Mg.ha-1), and Very High Yield (9.0 Mg.ha-1), considering all genotypes and regions. Our approach allows the prediction of environments (yield clusters) for a diverse set of growing conditions and revealed geographic and climatic causes of environmental quality, which differ according to each region and genotype group. From the LMM analysis, we found that the current relation between genetics (G), environmental variation (E), and GE for rainfed rice in Brazil is 1:6:2, but when we introduced our data-driven clusters (ME), the ratio decreased to 1:5:1. Consequently, the selection reliability for local adaptability across an extensive region increases. Our approach helps to identify mega-environments in Brazil that could be used as a target population of environments (TPE) of breeding programs. Additionally, it helps to identify more productive and stable seed production fields. HighlightsO_LIA nationwide environmental characterization and its relation to the genotype by environment interaction (GE) for grain yield of rainfed rice growing regions in Brazil. C_LIO_LIA data-driven approach capable to identifying clusters of yield levels and a machine learning approach to relate those clusters with environmental typologies. C_LIO_LIUnrevealed geographic and climatic causes of environmental quality for a group of genotypes or cultivar-specific predictions. C_LIO_LIThe strategy benefits diverse stages of breeding (multiple environmental trial analysis) and post-breeding (selection of fields for seed production) as an alternative approach to reduce costs and support decisions on cultivar planting locations. C_LI

genetics↗

bioRxiv2019

Functional arrays of human pluripotent stem cell-derived cardiac microtissues

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be amenable to standard multiwell-plate manipulations and be capable of quantifying tissue-level functions such as contractile force. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom printed multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a ramp. The wells are seeded with cell-laden collagen which, in response to the slope of the ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells and we used it to determine an optimal tissue formulation. We validated contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

bioengineering↗