Protocol for Isolation of Cardiac Interstitial Cells from Adult Murine Hearts for Unbiased Single Cell Profiling.

Interstitial cells have a crucial role in cardiac fibrosis and repair of the mammalian heart. Single-cell profiling using droplet-based technology has revolutionized the investigation of cell states and identities. Here, we present a protocol for the efficient isolation of high-quality live nucleated non-cardiomyocytes from adult murine heart, for unbiased single-cell RNA sequencing using 10× Chromium technology. This protocol has been applied to homeostatic and injured hearts from different mouse strains. For complete details on the use and execution of this protocol, please refer to Forte et al. (2020)

Potential biological meaning and origin of cardiac progenitor cells isolated as Cardiospheres

Several populations of progenitor cells have been identified in the adult heart, based on the expression of specific surface markers (c-kit, Sca1), or on functional properties, such as the ability to spontaneously migrate from tissue explants and grow in three dimensional structures, called cardiospheres (CSs). CSs represent a niche-like microtissue with a cardiogenic gradient of differentiation towards the periphery. It is not clear which is the origin of these endogenous progenitor populations and how they function in vivo, given the limited regenerative capacity of the heart. Several possibilities can be envisioned. These cells could be remnants of the embryonic development, or derive from the de-differentiation of cardiomyocytes. To test this hypothesis we used a double transgenic mouse expressing the recombinase MerCreMer under the cardiomyocyte-specific αMyosin-Heavy-Chain promoter. After a pulse with 4OH-tamoxifen (TAM) the activated Cre removes a stop signal between two LoxP sequences, allowing the expression of the reporter gene LacZ, in a spatial and temporal regulated manner. Ideally with this system all the differentiated cardiomyocytes should be irreversibly labeled after TAM treatment, even if they subsequently undergo de-differentiation. B-gal positive cells were very rare in culture, as shown by x-gal staining and real time PCR, suggesting that cardiomyocytes de-differentiation is not the main mechanism underlying CSs formation. However, given the limited sensibility of this method (the efficiency of recombination in the cardiomyocytes is 80% at maximum), we cannot conclusively exclude the occurrence of this phenomenon. Using another transgenic mouse strain, in which epicardial and epicardial-derived cells are labeled, we observed a large number of positive cells at all culture stages. Our hypothesis, supported by RT-PCR data, is that epicardial-derived cells undergo mesenchyma-to-epithelium (MET) transition during cardiospheres formation. This is consistent with a recent report presenting MET as a crucial mechanism in cell reprogramming

EMT/MET at the crossroad of stemness, regeneration and oncogenesis. The Ying-Yang equilibrium recapitulated in cell spheroids

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target

Ex uno, plures-From One Tissue to Many Cells: A Review of Single-Cell Transcriptomics in Cardiovascular Biology.

Recent technological advances have revolutionized the study of tissue biology and garnered a greater appreciation for tissue complexity. In order to understand cardiac development, heart tissue homeostasis, and the effects of stress and injury on the cardiovascular system, it is essential to characterize the heart at high cellular resolution. Single-cell profiling provides a more precise definition of tissue composition, cell differentiation trajectories, and intercellular communication, compared to classical bulk approaches. Here, we aim to review how recent single-cell multi-omic studies have changed our understanding of cell dynamics during cardiac development, and in the healthy and diseased adult myocardium

New perspectives to repair a broken heart

The aim of cardiac cell therapy is to restore at least in part the functionality of the diseased or injured myocardium by the use of stem/ progenitor cells. Recent clinical trials have shown the safety of cardiac cell therapy and encouraging efficacy results. A surprisingly wide range of non-myogenic cell types improves ventricular function, suggesting that benefits may result in part from mechanisms that are distinct from true myocardial regeneration. While clinical trials explore cells derived from skeletal muscle and bone marrow, basic researchers are investigating sources of new cardiomyogenic cells, such as resident myocardial progenitors and embryonic stem cells. In this commentary we briefly review the evolution of cell-based cardiac repair, some progress that has been made toward this goal, and future perspectives in the regeneration of cardiac tissue. © 2009 Bentham Science Publishers Ltd

Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart

Experimental data suggest that cell-based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit+ CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild-type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate 'cardiospheres', a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit+ CSCs

NURSING’S RELATIONSHIP WITH MEDICATION ERRORS: AN INTEGRATIVE REVIEW

Revisão integrativa com o objetivo de identificar na literatura a relação dos erros de medicação com a equipe de enfermagem. A pesquisa foi realizada por duas colaboradoras independentes, em três bases de dados, no período 2011 a 2015, com 32 estudos. Os resultados foram divididos em duas macrocategorias: características dos estudos e as relações diretas e indiretas da enfermagem com os erros de medicação. As relações diretas se referem às questões do âmbito profissional, como a falta de conhecimento e de experiência, falhas na comunicação, estresse e distração dos profissionais. As relações indiretas são compostas por questões de prescrição e dispensação, as condições físicas do paciente e erros na produção dos medicamentos. Conclui-se que, além de a enfermagem atuar na administração de medicamentos com vistas à assistência segura ao paciente, pode atuar como barreira dos erros provenientes de outras fases do processo de medicação.This integrative review aimed to identify in the literature the relationship between medication errors and the nursing team. The study was undertaken by two independent collaborators, in three databases, in the period 2011 – 2015, with 32 studies. The results were divided in two macrocategories: characteristics of the studies, and the direct and indirect relationships between nursing and the medication errors. The direct relationships refer to issues of the professional ambit, such as lack of knowledge and of experience, failures in communication, stress and the distraction of the professionals. The indirect relationships are made up of issues related to prescribing and dispensing, the patient’s physical conditions, and errors in the production of the medications. It is concluded that, besides the nursing staff working in administering medications with a view to safe care for the patient, they can act as a barrier to errors arising from other phases of the medication process.Revisión integrativa cuyo objetivo fue identificar, en la literatura, la relación de los errores de medicación con el equipo de enfermería. Se realizó la investigación por dos colaboradoras independientes, en tres bases de datos, en el periodo de 2011 a 2015, con 32 estudios. Los resultados fueron organizados en dos macrocategorías: características de los estudios y las relaciones directas y indirectas de la enfermería con los errores de medicación. Las relaciones directas se refieren a las cuestiones del ámbito profesional, como la falta de conocimiento y de experiencia, faltas en la comunicación, estrés y distracción de los profesionales. Las relaciones indirectas se constituyen por cuestiones de prescripción y dispensación, las condiciones físicas del paciente y errores en la producción de los medicamentos. Se concluye que, además de la enfermería actuar en la administración de medicamentos para una asistencia segura al paciente, puede actuar como barrera de los errores provenientes de otras fases del proceso de medicación

Serum and supplement optimization for EU GMP-compliance in cardiospheres cell culture

Cardiac progenitor cells (CPCs) isolated as cardiospheres (CSs) and CS-derived cells (CDCs) are a promising tool for cardiac cell therapy in heart failure patients, having CDCs already been used in a phase I/II clinical trial. Culture standardization according to Good Manufacturing Practices (GMPs) is a mandatory step for clinical translation. One of the main issues raised is the use of xenogenic additives (e.g. FBS, foetal bovine serum) in cell culture media, which carries the risk of contamination with infectious viral/prion agents, and the possible induction of immunizing effects in the final recipient. In this study, B27 supplement and sera requirements to comply with European GMPs were investigated in CSs and CDCs cultures, in terms of process yield/efficiency and final cell product gene expression levels, as well as phenotype. B27- free CS cultures produced a significantly reduced yield and a 10-fold drop in c-kit expression levels versus B27+ media. Moreover, autologous human serum (aHS) and two different commercially available GMP AB HSs were compared with standard research-grade FBS. CPCs from all HSs explants had reduced growth rate, assumed a senescent-like morphology with time in culture, and/or displayed a significant shift towards the endothelial phenotype. Among three different GMP gamma-irradiated FBSs (giFBSs) tested, two provided unsatisfactory cell yields, while one performed optimally, in terms of CPCs yield/phenotype. In conclusion, the use of HSs for the isolation and expansion of CSs/CDCs has to be excluded because of altered proliferation and/or commitment, while media supplemented with B27 and the selected giFBS allows successful EU GMP-complying CPCs culture