Biology subjects

Costa, K. D.

Publications and source records attributed to Costa, K. D..

6 recordsLinked to original sources

bioRxiv2024

Synergistic generation of cardiac resident-like macrophages and cardiomyocyte maturation in tissue engineered platforms

Cardiovascular disease stands as the leading cause of death globally, claiming approximately 19million lives in 2020. On the contrary, the development of cardiovascular drugs is experiencing a decline, largely due to the bottleneck in understanding the pathophysiology of various heart diseases and assessing the effects of drugs on healthy human hearts. The development of induced pluripotent stem cell (iPSC) technology and the availability of cardiac cell types in vitro, has resulted in a surge in efforts to fabricate human cardiac models for disease modelling and drug discovery applications. Although numerous attempts evidence successful fabrication of 3 dimensional (3D) engineered heart tissues, the innate immune cell population of the myocardium - particularly cardiac macrophages, was until recently, overlooke. With increasing appreciation of the interactions between cardiomyocytes and macrophages in the myocardium, in this work, isogenic populations of cardiac resident-like macrophages and cardiomyocytes were generated using iPSCs, to understand the interactions between the two cell types in both 2D and 3D settings, and subjected to electric stimulation. After characterizing iPSC-derived macrophages (iMacs) and iPSC-derived cardiomyocytes (iCMs) in depth, the conditioning of iMacs to align to a cardiac resident macrophage-like phenotype in the presence of iCMs in 2D culture was explored. In co-culture with iCMs, iMacs upregulated known genes expressed by cardiac resident macrophages. Additionally, in co-culture with iMacs, iCMs displayed an elongated morphology, improved calcium function and an increase in known maturation genes such as the ratio between MYH7 and MYH6 as well as SERCA2. In a 2D setting, iMacs showed the ability to electrically couple with iCMs and facilitate synchronous beating in iCM cultures. The 2D characterisation was translated into an engineered cardiac tissue model, wherein, improvement in tissue characteristics in the presence of iMacs was demonstrated in terms of increased cell alignment, enhanced cardiomyocyte elongation, physiologically relevant beat rates and improved tissue compaction. Taken together, these findings may open new avenues to use iMacs in engineered cardiac tissue models, not only as an innate immune cell source, but also as a support cell type to improve cardiomyocyte function and maturation.

bioengineering↗

bioRxiv2024

Reversal of contractile defects by mediating calcium homeostasis in human mini-heart models of heart failure with preserved ejection fraction (HFpEF) leads to first-in-human gene therapy clinical trial

AimsHeart failure with preserved ejection fraction (HFpEF), is a global health problem lacking disease-modifying therapeutic options, reflecting a lack of predictive models for preclinical drug testing. Aligned with FDA Modernization Act 2.0, we aimed to create the first in vitro human-specific mini-heart models of HFpEF, and to test the efficacy of a candidate gene therapy to improve cardiac kinetics and correct the disease phenotype. Methods and ResultsHealthy human pluripotent stem cell-derived ventricular cardiomyocytes were used to bioengineer beating cardiac tissue strips and pumping cardiac chambers. When conditioned with transforming growth factor-{beta}1 and endothelin-1, these mini-heart models exhibited signature disease phenotypes of significantly elevated diastolic force and tissue stiffness, and slowed contraction and relaxation kinetics, with no significant deficit in systolic force or ejection fraction versus unconditioned controls. Bioinformatic analysis of bulk RNA sequencing data from HFpEF mini-heart models and patient ventricular samples identified downregulation of SERCA2a of the calcium signalling pathway as a key differentially expressed gene. After dosage optimization, AAV-mediated expression of SERCA2a abrogated the disease phenotype and improved the cardiac kinetics in HFpEF mini-Hearts. ConclusionsThese findings contributed to FDA approval of an ongoing first-in-human gene therapy clinical trial for HFpEF, with Fast Track designation. We conclude that such human-based disease-specific mini-heart platforms are relevant for target discovery and validation that can facilitate clinical translation of novel cardiac therapies. Translational PerspectiveHeart failure with preserved ejection fraction (HFpEF) is a significant and growing global health concern lacking disease-modifying therapeutic options, reflecting inadequate preclinical models of the disease. Aligned with FDA Modernization Act 2.0, we created the first in vitro human-specific mini-heart models of HFpEF, demonstrated phenotypic disease characteristics of elevated stiffness and slowed kinetics, showed transcriptomic consistency with HFpEF patient data, identified SERCA2a as a key downregulated gene, performed dosing titration of SERCA2a gene therapy, and showed improvement of cardiac kinetics post-treatment. The findings contributed to FDA approval of an ongoing first-in-human gene therapy clinical trial for HFpEF.

bioengineering↗

bioRxiv2023

Auxetic patch material exhibits systolic thickening and restores pump function in a finite element model of acute myocardial infarction repair

Passive mechanical reinforcement of the infarcted heart has been shown to counteract infarct expansion and left ventricular (LV) functional degradation. However, traditional patch plasty of the ischemic region also restricts diastolic filling, reducing cardiac output. These negative side-effects can be minimized with strategic modification of the standard patch, suggesting further functional improvements could be possible through a broader exploration of patch materials. This study examines the potential advantages of a patch graft with auxetic properties, having a negative Poissons ratio ({nu} < 0). For preliminary evaluation, an established finite element model of LV biomechanics pre- and post-acute infarction, originally developed for modeling patch plasty with non-auxetic, or meiotic, materials, was modified to simulate epicardial implantation of auxetic patch materials. A homogeneous auxetic patch graft ({nu} = -0.2) exhibited radial thickening during systole, driven by tension on the patch from the neighboring contracting myocardium; but the patch thickness expanded away from the LV cavity and therefore did not improve ventricular mechanics compared to a standard meiotic patch graft ({nu} = 0.4). Alternatively, a heterogeneous auxetic patch design with an outer reinforcement layer caused inwardly-directed systolic thickening that restored the LV pressure-volume relationship toward baseline function, without adversely affecting fiber stress in the border zone or remote myocardium compared to the meiotic patch. This computational modeling study demonstrates the potential to harness auxetic mechanical properties for improving LV pump function in the setting of acute myocardial infarction, motivating further experimental validation of auxetic metamaterial patch devices for surgical repair of injured myocardium.

bioengineering↗

bioRxiv2023

Robotic System for Organoid Assembly in a Multi-Well Microfluidic Chip

While many cell culture systems are sensitive to the conditions in which cells are introduced into the system, we find that in situ differentiated tube-shaped microfluidic organoids have a particularly high sensitivity. Preliminary experiments using conventional seeding techniques revealed that biases in initial cell number and distribution dramatically impacted organoid shape and behavior downstream. Residual flows during seeding further complicated the process, dispersing cells to undesirable locations within the chip. To address this problem, a a robotic seeding system for controlling the process of inserting cells into microfluidic chips was developed. Environmental control of temperature, CO2, and humidity was implemented by modifying a commercial Arduino-controlled incubator. An eight-channel syringe pump controlled flow to eight cell dispensers, while a vertical leadscrew stage raised and lowered them, and a set of stackable flexure micromanipulators individually controlled the X and Y position of each cell dispenser. The flexure manipulators were 3D printed, driven by low-cost motors and electronics, and required little assembly and no alignment, resulting in a cheap and scalable method of controlling a dense array of micromanipulators. A dual objective microscope on a motorized gantry used an oblique lighting system to observe the seeding process, allowing for real-time interventions or passive observation of automated protocols. The robotic cell seeding system provided a platform for optimizing a sensitive process towards increasing the repeatability and physiological relevance of tube-shaped microfluidic organoids.

bioengineering↗

bioRxiv2023

Automated Stimulation and Long-Term Remote Monitoring of Multi-Plex Microfluidic Organoid Chips

Developing a novel microfluidic organoid system required many experiments and iterations due to lack of knowledge about the relevant developmental biology. Collecting data on the developing organoids quickly escalated into a bottleneck as high throughput and long term culture resulted in a rapidly increasing number of specimens being observed. Commercially available automated microscope systems exist, but were either too expensive or not appropriate, and could not be modified. To satisfy the increasing need for automated data collection, a custom robotic system was developed to collect data from within a standard incubator. An X-Y belt driven gantry was designed with an architecture chosen to balance high accuracy, low cost, speed, and range of motion. Focus control was implemented with dual miniature leadscrews. A linear sliding mechanism was used to switch between two microscope objectives. 3D printed chip attachments were designed to implement illuminators for bright field imaging, and electrodes for stimulating the cardiac organoids. A fluorescent filter block was designed using a 3D printed piece to hold optical components, and a multi-band filter set that allowed for three color fluorescence without moving parts. A pulley driven tilting stage gravitationally biased the organoids during development. In order to ensure accurate image collection despite the inevitable position shifting of the chips, an image processing pipeline was developed for locating organoids using geometrical microfluidic chip features. The resulting robotic system automated imaging data collection on organoids and electrical and mechanical stimulation, in addition to being modifiable for future projects.

bioengineering↗

bioRxiv2023

Intrinsic Mechanics of Human Stem Cell Derived Aortic Smooth Muscle Cells Support a Developmental Basis for Aneurysm Localization in Marfan Syndrome

Marfan Syndrome (MFS), a connective tissue disorder caused by a mutation in the fibrillin-1 gene, occurs in approximately 1 in 5,000 people worldwide. As an important constituent of the extracellular matrix, mutated fibrillin-1 in Marfan Syndrome leads to aortic medial degeneration, aneurysm, and dissection. TGF{beta} in the matrix, which is controlled by fibrillin-1, is known to cause pathological effects in smooth muscle cells (SMCs) within the aortic wall during MFS. TGF{beta} as well as other cytokines have been shown to impact neural crest derived SMCs differently than mesodermal derived SMCs. Furthermore, outcomes of variable cytokine responsiveness of neural crest SMCs are compounded by genetically imposed changes to neural crest SMC integrin distributions in MFS. Thus, it has been hypothesized that neural crest derived SMCs, which give rise to ascending aortic SMCs, are intrinsically mechanically susceptible to aneurysm formation in MFS. This hypothesis has been linked to the clinical observation of aneurysm formation preferentially occurring in the ascending versus descending aorta in MFS. We aim to test the hypothesis that aortic smooth muscle cells (ASMCs) have intrinsic mechanobiological properties which cause cell weakening in Marfan Syndrome. Human induced pluripotent stem cells (hiPSC) from Marfan patients and healthy volunteers were differentiated into either ascending- or descending-ASMCs via their respective developmental lineages, and cultured to either an early (6 days) or late (30 days) stage of post-differentiation maturation. Mass spectrometry-based proteomics of early-stage iPSC-ASMCs revealed an array of depleted proteins unique to MFS ascending-SMCs that were associated with cell mechanics and aortic aneurysm. Targeted examination of the proteomics dataset revealed intracellular proteins (ACTA2, CNN1, TAGLN) were significantly depleted in MFS ascending-ASMCs. The intrinsic, matrix-independent, hiPSC-ASMC stiffness quantified by atomic force microscopy (AFM) revealed that MFS ascending-ASMCs, but not descending-ASMCs, were significantly less stiff than healthy, at the late cell-maturation stage (p<0.0005). Late-stage ascending- and descending-ASMCs also showed clear functional impairments via calcium flux in MFS. AFM revealed a similar mechanical phenotype in early-stage ASMCs, with MFS ascending-ASMCs, but not descending-ASMCs, being significantly less stiff than healthy (p<0.005). In summary, this study supports an emerging hypothesis of ontogenetic predisposition for aneurysm susceptibility in Marfan Syndrome based on locally altered mechanobiology of developmental origin-specific ASMC subtypes. This may lead to new cell-targeted approaches for treating aortic aneurysm in patients with MFS.

biophysics↗