3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes.

Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13 C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs.

Unveiling Human Proteome Signatures of Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent but still poorly understood clinical entity. Its current pathophysiological understanding supports a critical role of comorbidities and their chronic effect on cardiac function and structure. Importantly, despite the replication of some HFpEF phenotypic features, to this day, experimental models have failed to bring new effective therapies to the clinical setting. Thus, the direct investigation of HFpEF human myocardial samples may unveil key, and possibly human-specific, pathophysiological mechanisms. This study employed quantitative proteomic analysis by advanced mass spectrometry (SWATH-MS) to investigate signaling pathways and pathophysiological mechanisms in HFpEF. Protein-expression profiles were analyzed in human left ventricular myocardial samples of HFpEF patients and compared with a mixed control group. Functional analysis revealed several proteins that correlate with HFpEF, including those associated with mitochondrial dysfunction, oxidative stress, and inflammation. Despite the known disease heterogeneity, proteomic profiles could indicate a reduced mitochondrial oxidative phosphorylation and fatty-acid oxidation capacity in HFpEF patients with diabetes. The proteomic characterization described in this work provides new insights. Furthermore, it fosters further questions related to HFpEF cellular pathophysiology, paving the way for additional studies focused on developing novel therapies and diagnosis strategies for HFpEF patients.

Stem cells characterization: OMICS reinforcing analytics.

Stem cells hold outstanding potential to model and treat disease and are valuable tools in pharmacology and toxicology. Characterization of stem cells and derivatives still poses many challenges to ensure safe, efficacious, and reliable therapies. Regulatory agencies have defined key mandatory attributes related to identity, purity, sterility, and genomic integrity, however robust analytics to determine cell's potency are still a major challenge, in most cases assessed case-by-case. Importantly, the application of high-throughput 'omic tools is opening new perspectives on stem cell's research and development. Here, analytical methodologies currently employed to characterize stem cells' quality attributes are discussed, with special focus on 'omics as relevant tools for definition of cell's mechanism of action, and for potency assay development and assessment.

Proteomic and Glyco(proteo)mic tools in the profiling of cardiac progenitors and pluripotent stem cell derived cardiomyocytes: Accelerating translation into therapy.

Research in stem cells paved the way to an enormous amount of knowledge, increasing expectations on cardio regenerative therapeutic approaches in clinic. While the first generation of clinical trials using cell-based therapies in the heart were performed with bone marrow and adipose tissue derived mesenchymal stem cells, second generation cell therapies moved towards the use of cardiac-committed cell populations, including cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes. Despite all these progresses, translating the aptitudes of R&D and pre-clinical data into effective clinical treatments is still highly challenging, partially due to the demanding regulatory and safety concerns but also because of the lack of knowledge on the regenerative mechanisms of action of these therapeutic products. Thus, the need of analytical methodologies that enable a complete characterization of such complex products and a deep understanding of their therapeutic effects, at the cell and molecular level, is imperative to overcome the hurdles of these advanced therapies. Omics technologies, such as proteomics and glyco(proteo)mics workflows based on state of the art mass-spectrometry, have prompted some major breakthroughs, providing novel data on cell biology and a detailed assessment of cell based-products applied in cardiac regeneration strategies. These advanced 'omics approaches, focused on the profiling of protein and glycan signatures are excelling the identification and characterization of cell populations under study, namely unveiling pluripotency and differentiation markers, as well as paracrine mechanisms and signaling cascades involved in cardiac repair. The leading knowledge generated is supporting a more rational therapy design and the rethinking of challenges in Advanced Therapy Medicinal Products development. Herein, we review the most recent methodologies used in the fields of proteomics, glycoproteomics and glycomics and discuss their impact on the study of cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes biology. How these discoveries will impact the speed up of novel therapies for cardiovascular diseases is also addressed.

Bioreactor-based 3D human myocardial ischemia/reperfusion in vitro model: a novel tool to unveil key paracrine factors upon acute myocardial infarction.

During acute myocardial infarction (AMI), Ischemia/Reperfusion (I/R) injury causes cardiomyocyte (CM) death and loss of tissue function, making AMI one of the major causes of death worldwide. Cell-based in vitro models of I/R injury have been increasingly used as a complementary approach to preclinical research. However, most approaches use murine cells in 2D culture setups, which are not able to recapitulate human cellular physiology, as well as nutrient and gas gradients occurring in the myocardium. In this work we established a novel human in vitro model of myocardial I/R injury using CMs derived from human induced pluripotent stem cells (hiPSC-CMs), which were cultured as 3D aggregates in stirred tank bioreactors. We were able to recapitulate important hallmarks of AMI, including loss of CM viability with disruption of cellular ultrastructure, increased angiogenic potential, and secretion of key proangiogenic and proinflammatory cytokines. Conditioned medium was further used to probe human cardiac progenitor cells (hCPCs) response to paracrine cues from injured hiPSC-CMs through quantitative whole proteome analysis (SWATH-MS). I/R injury hiPSC-CM conditioned media incubation caused upregulation of hCPC proteins associated with migration, proliferation, paracrine signaling, and stress response-related pathways, when compared to the control media incubation. Our results indicate that the model developed herein can serve as a novel tool to interrogate mechanisms of action of human cardiac populations upon AMI.

Human cardiac progenitor cell activation and regeneration mechanisms: exploring a novel myocardial ischemia/reperfusion in vitro model.

BACKGROUND: Numerous studies from different labs around the world report human cardiac progenitor cells (hCPCs) as having a role in myocardial repair upon ischemia/reperfusion (I/R) injury, mainly through auto/paracrine signaling. Even though these cell populations are already being investigated in cell transplantation-based clinical trials, the mechanisms underlying their response are still poorly understood. METHODS: To further investigate hCPC regenerative process, we established the first in vitro human heterotypic model of myocardial I/R injury using hCPCs and human-induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs). The co-culture model was established using transwell inserts and evaluated in both ischemia and reperfusion phases regarding secretion of key cytokines, hiPSC-CM viability, and hCPC proliferation. hCPC proteome in response to I/R was further characterized using advanced liquid chromatography mass spectrometry tools. RESULTS: This model recapitulates hallmarks of I/R, namely hiPSC-CM death upon insult, protective effect of hCPCs on hiPSC-CM viability (37.6% higher vs hiPSC-CM mono-culture), and hCPC proliferation (approximately threefold increase vs hCPCs mono-culture), emphasizing the importance of paracrine communication between these two populations. In particular, in co-culture supernatant upon injury, we report higher angiogenic functionality as well as a significant increase in the CXCL6 secretion rate, suggesting an important role of this chemokine in myocardial regeneration. hCPC whole proteome analysis allowed us to propose new pathways in the hCPC-mediated regenerative process, including cell cycle regulation, proliferation through EGF signaling, and reactive oxygen species detoxification. CONCLUSION: This work contributes with new insights into hCPC biology in response to I/R, and the model established constitutes an important tool to study the molecular mechanisms involved in the myocardial regenerative process.

Human cardiac stem cells inhibit lymphocyte proliferation through paracrine mechanisms that correlate with indoleamine 2,3-dioxygenase induction and activity.

Transplantation of allogeneic human cardiac/stem progenitor cells (hCSCs) is currently being tested in several phase I/II clinical trials as a novel and promising therapy for restoration of myocardial tissue function in acute myocardial infarction (AMI) patients. Previous findings demonstrate that these cells have an immune suppressive profile interacting with different populations from the immune system, resulting in overall attenuation of myocardial inflammation. However, transplanted hCSCs are still recognized and cleared from the injured site, impairing long retention times in the tissue that could translate into a higher clinical benefit.In this work, through modeling allogeneic hCSC/T lymphocyte interaction in vitro by direct contact, transwell inserts, and hCSC conditioned medium, our results demonstrate that hCSCs exert an immune-suppressive effect on T lymphocyte proliferation not only through the previously described cell contact-dependent programmed cell death-1 (PD1)/programmed death ligand-1 (PDL-1) axis but also through a paracrine mechanism associated with indoleamine 2,3-dioxygenase (IDO) enzyme-mediated tryptophan metabolism. Such findings constitute a step forward in better understanding the mechanisms of action of transplanted hCSCs in allogeneic settings.