The Genetics of Human Congenital Coronary Vascular Anomalies.

The genetics of human congenital coronary vascular anomalies (hCCVA) remains largely underresearched. This is surprising, because although coronary vascular defects represent a relatively small proportion of human congenital heart disease (CHD), hCCVAs are clinically significant conditions. Indeed, hCCVA frequently associate to other congenital cardiac structural defects and may even result in sudden cardiac death in the adult. In this brief chapter, we will attempt to summarize our current knowledge on the topic, also proposing a rationale for the development of novel approaches to the genetics of hCCVA.

Congenital Coronary Blood Vessel Anomalies: Animal Models and the Integration of Developmental Mechanisms.

Coronary blood vessels are in charge of sustaining cardiac homeostasis. It is thus logical that coronary congenital anomalies (CCA) directly or indirectly associate with multiple cardiac conditions, including sudden death. The coronary vascular system is a sophisticated, highly patterned anatomical entity, and therefore a wide range of congenital malformations of the coronary vasculature have been described. Despite the clinical interest of CCA, very few attempts have been made to relate specific embryonic developmental mechanisms to the congenital anomalies of these blood vessels. This is so because developmental data on the morphogenesis of the coronary vascular system derive from complex studies carried out in animals (mostly transgenic mice), and are not often accessible to the clinician, who, in turn, possesses essential information on the significance of CCA. During the last decade, advances in our understanding of normal embryonic development of coronary blood vessels have provided insight into the cellular and molecular mechanisms underlying coronary arteries anomalies. These findings are the base for our attempt to offer plausible embryological explanations to a variety of CCA as based on the analysis of multiple animal models for the study of cardiac embryogenesis, and present them in an organized manner, offering to the reader developmental mechanistic explanations for the pathogenesis of these anomalies.

Corrigendum: NK cell subsets changes in partial remission and early stages of pediatric type 1 diabetes.

[This corrects the article DOI: 10.3389/fimmu.2020.611522.].

Epicardium and Coronary Vessels.

Congenital anomalies and acquired diseases of the coronary blood vessels are of great clinical relevance. The early diagnosis of these conditions remains, however, challenging. In order to improve our knowledge of these ailments, progress has to be achieved in the research of the molecular and cellular mechanisms that control development of the coronary vascular bed. The aim of this chapter is to provide a succint account of the key elements of coronary blood vessel development, especially in the context of the role played by the epicardium and epicardial cellular derivatives. We will discuss the importance of the epicardium in coronary blood vessel morphogenesis, from the contribution of the epicardially derived mesenchyme to these blood vessels to its role as an instructive signaling center, attempting to relate these concepts to the origin of coronary disease.

Artificial Gametes and Human Reproduction in the 21st Century: An Ethical Analysis.

Artificial gametes, derived from stem cells, have the potential to enable in vitro fertilization of embryos. Currently, artificial gametes are only being generated in laboratory animals; however, considerable efforts are underway to develop artificial gametes using human cell sources. These artificial gametes are being proposed as a means to address infertility through assisted reproductive technologies. Nonetheless, the availability of artificial gametes obtained from adult organisms can potentially expand the possibilities of reproduction. Various groups, such as same-sex couples, post-menopausal women, and deceased donors, could potentially utilize artificial gametes to conceive genetically related offspring. The advent of artificial gametes raises significant bioethical questions. Should all these reproductive scenarios be accepted? How can we delineate the range of future reproductive choices? A normative bioethical framework may be necessary to establish a consensus regarding the use of human artificial gametes. This review aims to present the current state of research on the biological roadmap for generating artificial gametes, while also summarizing proposed approaches to establish a normative framework that delineates ethically acceptable paths for reproduction.

A 3D atlas of the human developing pancreas to explore progenitor proliferation and differentiation.

AIMS/HYPOTHESIS: Rodent pancreas development has been described in great detail. On the other hand, there are still gaps in our understanding of the developmental trajectories of pancreatic cells during human ontogenesis. Here, our aim was to map the spatial and chronological dynamics of human pancreatic cell differentiation and proliferation by using 3D imaging of cleared human embryonic and fetal pancreases. METHODS: We combined tissue clearing with light-sheet fluorescence imaging in human embryonic and fetal pancreases during the first trimester of pregnancy. In addition, we validated an explant culture system enabling in vitro proliferation of pancreatic progenitors to determine the mitogenic effect of candidate molecules. RESULTS: We detected the first insulin-positive cells as early as five post-conceptional weeks, two weeks earlier than previously observed. We observed few insulin-positive clusters at five post-conceptional weeks (mean ± SD 9.25±5.65) with a sharp increase to 11 post-conceptional weeks (4307±152.34). We identified a central niche as the location of onset of the earliest insulin cell production and detected extra-pancreatic loci within the adjacent developing gut. Conversely, proliferating pancreatic progenitors were located in the periphery of the epithelium, suggesting the existence of two separated pancreatic niches for differentiation and proliferation. Additionally, we observed that the proliferation ratio of progenitors ranged between 20% and 30%, while for insulin-positive cells it was 1%. We next unveiled a mitogenic effect of the platelet-derived growth factor AA isoform (PDGFAA) in progenitors acting through the pancreatic mesenchyme by increasing threefold the number of proliferating progenitors. CONCLUSIONS/INTERPRETATION: This work presents a first 3D atlas of the human developing pancreas, charting both endocrine and proliferating cells across early development.

Queering the genome: ethical challenges of epigenome editing in same-sex reproduction.

In this article, I explore the ethical dimensions of same-sex reproduction achieved through epigenome editing-an innovative and transformative technique. For the first time, I analyse the potential normativity of this disruptive approach for reproductive purposes, focusing on its implications for lesbian couples seeking genetically related offspring. Epigenome editing offers a compelling solution to the complex ethical challenges posed by traditional gene editing, as it sidesteps genome modifications and potential long-term genetic consequences. The focus of this article is to systematically analyse the bioethical issues related to the use of epigenome editing for same-sex reproduction. I critically assess the ethical acceptability of epigenome editing with reproductive purposes from multiple angles, considering harm perspectives, the comparison of ethical issues related to gene and epigenome editing, and feminist theories. This analysis reveals that epigenome editing emerges as an ethically acceptable means for lesbian couples to have genetically related children. Moreover, the experiments of a reproductive use of epigenome editing discussed in this article transcend bioethics, shedding light on the broader societal implications of same-sex reproduction. It challenges established notions of biological reproduction and prompts a reevaluation of how we define the human embryo, while poses some issues in the context of gender self-identification and family structures. In a world that increasingly values inclusivity and diversity, this article aims to reveal a progressive pathway for reproductive medicine and bioethics, as well as underscores the need for further philosophical research in this emerging and fertile domain.

Human stem-cell-derived embryo models: When bioethical normativity meets biological ontology.

The use of human stem-cell-derived embryo models in biomedical research has recently sparked intense bioethical debates. In this article, we delve into the ethical complexities surrounding these models and advocate for a deeper exploration of their biological ontology to discuss their bioethical normativity. We examine the ethical considerations arising from the implementation of these models, emphasizing varying viewpoints on their ethical standing and the ethical obligations associated with their development and utilization. We contend that a nuanced comprehension of their biological ontology is crucial for navigating these ethical quandaries. Furthermore, we underscore the indispensability of interdisciplinary cooperation among bioethicists, biologists, and philosophers to unravel the complex interplay between biological ontology and the normative framework of bioethics. Moreover, this article introduces a novel combinatorial approach to resolve the ethical dilemma surrounding these models. We propose a distinction between models that closely emulate natural embryos, based on the status of synthetic embryos, and those capable of reproducing specific dimensions of embryonic development. Such differentiation allows for nuanced ethical considerations while harnessing the value of these models in scientific research, paving the way for a more comprehensive ethical framework in the context of evolving biotechnologies.

Generation of heart and vascular system in rodents by blastocyst complementation.

Generating organs from stem cells through blastocyst complementation is a promising approach to meet the clinical need for transplants. In order to generate rejection-free organs, complementation of both parenchymal and vascular cells must be achieved, as endothelial cells play a key role in graft rejection. Here, we used a lineage-specific cell ablation system to produce mouse embryos unable to form both the cardiac and vascular systems. By mouse intraspecies blastocyst complementation, we rescued heart and vascular system development separately and in combination, obtaining complemented hearts with cardiomyocytes and endothelial cells of exogenous origin. Complemented chimeras were viable and reached adult stage, showing normal cardiac function and no signs of histopathological defects in the heart. Furthermore, we implemented the cell ablation system for rat-to-mouse blastocyst complementation, obtaining xenogeneic hearts whose cardiomyocytes were completely of rat origin. These results represent an advance in the experimentation towards the in vivo generation of transplantable organs.

The Ethics of Cellular Reprogramming.

Louise Brown's birth in 1978 heralded a new era not just in reproductive technology, but in the relationship between science, cells, and society. For the first time, human embryos could be created, selected, studied, manipulated, frozen, altered, or destroyed, outside the human body. But with this possibility came a plethora of ethical questions. Is it acceptable to destroy a human embryo for the purpose of research? Or to create an embryo with the specific purpose of destroying it for research? In an attempt to construct ethical and legal frameworks for the new era of cellular reprogramming, legislators and ethicists have tried to distinguish between different kinds of biological entity. We treat cells differently depending on whether they are human or animal, somatic cells or gametes, and on whether they are embryos or not. But this approach to the ethics of cellular reprogramming is doomed to failure for the simple reason that cellular reprogramming in itself destroys the distinctions that the law requires to function. In this article, we explore the historical trajectory of cellular reprogramming and its relationship with ethics and society. We suggest that the early hype of embryo research has not obviously fulfilled expectations, but since new avenues of research are continuously opening, it is hard to say definitely that these promises have been broken. We explore the forthcoming challenges posed by the creation of DNA from scratch in the laboratory, and the implications of this for understandings of identity, privacy, and reproduction. We conclude that while ethics used to seek answers in biological facts, this is no longer possible, and a new approach is required.

Synthetic embryos: a new venue in ethical research.

In brief: Two independent groups have reported the development of 'artificial embryos'. Those are in vitro models made of mouse embryonic stem cells, without the need for egg or sperm, and grown ex utero without requiring implantation. This system might open new venues in bioethical research if human cells show the ability to replicate this system. Abstract: The recent publications reported in 2022 reveal the possibility of obtaining mouse embryos without the need for egg or sperm. These 'artificial embryos' can recapitulate some stages of development ex utero - from neurulation to organogenesis - without implantation. Synthetic mouse embryos might serve as a valuable model to gain further insights into early developmental stages. Indeed, it is expected for these models to be replicated by employing human cells. This promising research raises ethical issues and expands the horizon of ethics in regard to the development of the human embryo. From this point of view, we state some of the new open venues for bioethical research.

Development of an injectable alginate-collagen hydrogel for cardiac delivery of extracellular vesicles.

Extracellular vesicles (EVs) are nanosized particles with attractive therapeutic potential for cardiac repair. However, low retention and stability after systemic administration limit their clinical translation. As an alternative, the combination of EVs with biomaterial-based hydrogels (HGs) is being investigated to increase their exposure in the myocardium and achieve an optimal therapeutic effect. In this study, we developed and characterized a novel injectable in-situ forming HG based on alginate and collagen as a cardiac delivery vehicle for EVs. Different concentrations of alginate and collagen crosslinked with calcium gluconate were tested. Based on injectability studies, 1% alginate, 0.5 mg/mL collagen and 0.25% calcium gluconate HG was selected as the idoneous combination for cardiac administration using catheter-based systems. Rheological examination revealed that the HG possessed an internal gel structure, weak mechanical properties and low viscosity, facilitating an easy administration. In addition, EVs were successfully incorporated and homogeneously distributed in the HG. After administration in a rat model of myocardial infarction, the HG showed long-term retention in the heart and allowed for a sustained release of EVs for at least 7 days. Thus, the combination of HGs and EVs represents a promising therapeutic strategy for myocardial repair. Besides EVs delivery, the developed HG could represent a useful platform for cardiac delivery of multiple therapeutic agents.

In Vivo and In Vitro Cartilage Differentiation from Embryonic Epicardial Progenitor Cells.

The presence of cartilage tissue in the embryonic and adult hearts of different vertebrate species is a well-recorded fact. However, while the embryonic neural crest has been historically considered as the main source of cardiac cartilage, recently reported results on the wide connective potential of epicardial lineage cells suggest they could also differentiate into chondrocytes. In this work, we describe the formation of cardiac cartilage clusters from proepicardial cells, both in vivo and in vitro. Our findings report, for the first time, cartilage formation from epicardial progenitor cells, and strongly support the concept of proepicardial cells as multipotent connective progenitors. These results are relevant to our understanding of cardiac cell complexity and the responses of cardiac connective tissues to pathologic stimuli.

Isolation methods of large and small extracellular vesicles derived from cardiovascular progenitors: A comparative study.

Since the discovery of the beneficial therapeutical effects of extracellular vesicles (EVs), these agents have been attracting great interest as next-generation therapies. EVs are nanosized membrane bodies secreted by all types of cells that mediate cell-cell communication. Although the classification of different subpopulations of EVs can be complex, they are broadly divided into microvesicles and exosomes based on their biogenesis and in large and small EVs based on their size. As this is an emerging field, current investigations are focused on basic aspects such as the more convenient method for EV isolation. In the present paper, we used cardiac progenitor cells (CPCs) to study and compare different cell culture conditions for EV isolation as well as two of the most commonly employed purification methods: ultracentrifugation (UC) and size-exclusion chromatography (SEC). Large and small EVs were separately analysed. We found that serum starvation of cells during the EV collecting period led to a dramatic decrease in EV secretion and major cell death. Regarding the isolation method, our findings suggest that UC and SEC gave similar EV recovery rates. Separation of large and small EV-enriched subpopulations was efficiently achieved with both purification protocols although certain difference in sample heterogeneity was observed. Noteworthy, while calnexin was abundant in large EVs, ALIX and CD63 were mainly found in small EVs. Finally, when the functionality of EVs was assessed on primary culture of adult murine cardiac fibroblasts, we found that EVs were taken up by these cells, which resulted in a pronounced reduction in the proliferative and migratory capacity of the cells. Specifically, a tendency towards a larger effect of SEC-related EVs was observed. No differences could be found between large and small EVs. Altogether, these results contribute to establish the basis for the use of EVs as therapeutic platforms, in particular in regenerative fields.

Pancreatic Islet Cells Response to IFNγ Relies on Their Spatial Location within an Islet.

Type 1 diabetes (T1D) is an auto-immune disease characterized by the progressive destruction of insulin-producing pancreatic beta cells. While beta cells are the target of the immune attack, the other islet endocrine cells, namely the alpha and delta cells, can also be affected by the inflammatory milieu. Here, using a flow cytometry-based strategy, we compared the impact of IFNγ, one of the main cytokines involved in T1D, on the three endocrine cell subsets isolated from C57BL/6 mouse islets. RNA-seq analyses revealed that alpha and delta cells exposed in vitro to IFNγ display a transcriptomic profile very similar to that of beta cells, with an increased expression of inflammation key genes such as MHC class I molecules, the CXCL10 chemokine and the programmed death-ligand 1 (PD-L1), three hallmarks of IFNγ signaling. Interestingly, at low IFNγ concentration, we observed two beta cell populations (responders and non-responders) based on PD-L1 protein expression. Our data indicate that this differential sensitivity relies on the location of the cells within the islet rather than on the existence of two different beta cells subsets. The same findings were corroborated by the in vivo analysis of pancreatic islets from the non-obese diabetic mouse model of T1D, showing more intense PD-L1 staining on endocrine cells close to immune infiltrate. Collectively, our work demonstrates that alpha and delta cells are as sensitive as beta cells to IFNγ, and suggests a gradual diffusion of the cytokine into an islet. These observations provide novel insights into the in situ inflammatory processes occurring in T1D progression.

Use and Misuse of Cq in qPCR Data Analysis and Reporting.

In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis. Because Cq is directly related to the starting concentration of the target, and the difference in Cq values is related to the starting concentration ratio, the only results of qPCR analysis reported are often Cq, ΔCq or ΔΔCq values. However, reporting of Cq values ignores the fact that Cq values may differ between runs and machines, and, therefore, cannot be compared between laboratories. Moreover, Cq values are highly dependent on the PCR efficiency, which differs between assays and may differ between samples. Interpreting reported Cq values, assuming a 100% efficient PCR, may lead to assumed gene expression ratios that are 100-fold off. This review describes how differences in quantification threshold setting, PCR efficiency, starting material, PCR artefacts, pipetting errors and sampling variation are at the origin of differences and variability in Cq values and discusses the limits to the interpretation of observed Cq values. These issues can be avoided by calculating efficiency-corrected starting concentrations per reaction. The reporting of gene expression ratios and fold difference between treatments can then easily be based on these starting concentrations.

Understanding the Adult Mammalian Heart at Single-Cell RNA-Seq Resolution.

During the last decade, extensive efforts have been made to comprehend cardiac cell genetic and functional diversity. Such knowledge allows for the definition of the cardiac cellular interactome as a reasonable strategy to increase our understanding of the normal and pathologic heart. Previous experimental approaches including cell lineage tracing, flow cytometry, and bulk RNA-Seq have often tackled the analysis of cardiac cell diversity as based on the assumption that cell types can be identified by the expression of a single gene. More recently, however, the emergence of single-cell RNA-Seq technology has led us to explore the diversity of individual cells, enabling the cardiovascular research community to redefine cardiac cell subpopulations and identify relevant ones, and even novel cell types, through their cell-specific transcriptomic signatures in an unbiased manner. These findings are changing our understanding of cell composition and in consequence the identification of potential therapeutic targets for different cardiac diseases. In this review, we provide an overview of the continuously changing cardiac cellular landscape, traveling from the pre-single-cell RNA-Seq times to the single cell-RNA-Seq revolution, and discuss the utilities and limitations of this technology.

Extracellular Vesicle-Based Therapeutics for Heart Repair.

Extracellular vesicles (EVs) are constituted by a group of heterogeneous membrane vesicles secreted by most cell types that play a crucial role in cell-cell communication. In recent years, EVs have been postulated as a relevant novel therapeutic option for cardiovascular diseases, including myocardial infarction (MI), partially outperforming cell therapy. EVs may present several desirable features, such as no tumorigenicity, low immunogenic potential, high stability, and fine cardiac reparative efficacy. Furthermore, the natural origin of EVs makes them exceptional vehicles for drug delivery. EVs may overcome many of the limitations associated with current drug delivery systems (DDS), as they can travel long distances in body fluids, cross biological barriers, and deliver their cargo to recipient cells, among others. Here, we provide an overview of the most recent discoveries regarding the therapeutic potential of EVs for addressing cardiac damage after MI. In addition, we review the use of bioengineered EVs for targeted cardiac delivery and present some recent advances for exploiting EVs as DDS. Finally, we also discuss some of the most crucial aspects that should be addressed before a widespread translation to the clinical arena.