An overview of international regulatory frameworks for mesenchymal stromal cell-based medicinal products: From laboratory to patient.

Human mesenchymal stromal cells (hMSCs) are emerging as one of the most important cell types in advanced therapies and regenerative medicine due to their great therapeutic potential. The development of hMSC-based products focuses on the use of hMSCs as biological active substances, and they are considered medicinal products by the primary health agencies worldwide. Due to their regulatory status, the development of hMSC-based products is regulated by specific criteria that range from the design phase, nonclinical studies, clinical studies, to the final registration and approval. Patients should only be administered hMSC-based products within the framework of a clinical trial or after the product has obtained marketing authorization; in both cases, authorization by health authorities is usually required. Considering the above, this paper describes the current general regulatory requirements for hMSC-based products, by jurisdiction, to be implemented throughout their entire development process. These measures may provide support for researchers from both public and private entities and academia to optimize the development of these products and their subsequent marketing, thereby improving access to them by patients.

Synthesis of Amino Terminal Clicked Dendrimers. Approaches to the Application as a Biomarker.

Herein, we present an easy and efficient synthesis of amino terminal dendrons, combining protection/deprotection reactions with copper-catalyzed azide alkyne cycloaddition in a convergent way. This new approach affords dendrons in gram scale with excellent yields and easy purification. By choosing the appropriate azido-functionalized core, these dendrons lead to a more efficient and controlled convergent synthesis of dendrimers with different sizes and shapes and multivalence. The amino terminal dendrimers were analyzed by diffusion-ordered spectroscopy experiments. The observed dendrimer size is in excellent correlation with the expected size and shape by molecular dynamic simulations. The construction of these kinds of nanostructures, in a simple and efficient way, opens new opportunities for biomedical applications. Moreover, by choosing the appropriate core, these versatile macromolecules become an excellent fluorescent biomarker.

Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the ß-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.

A New Versatile Platform for Assessment of Improved Cardiac Performance in Human-Engineered Heart Tissues.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) hold a great potential as human in vitro models for studying heart disease and for drug safety screening. Nevertheless, their associated immaturity relative to the adult myocardium limits their utility in cardiac research. In this study, we describe the development of a platform for generating three-dimensional engineered heart tissues (EHTs) from hPSC-CMs for the measurement of force while under mechanical and electrical stimulation. The modular and versatile EHT platform presented here allows for the formation of three tissues per well in a 12-well plate format, resulting in 36 tissues per plate. We compared the functional performance of EHTs and their histology in three different media and demonstrated that tissues cultured and maintained in maturation medium, containing triiodothyronine (T3), dexamethasone, and insulin-like growth factor-1 (TDI), resulted in a higher force of contraction, sarcomeric organization and alignment, and a higher and lower inotropic response to isoproterenol and nifedipine, respectively. Moreover, in this study, we highlight the importance of integrating a serum-free maturation medium in the EHT platform, making it a suitable tool for cardiovascular research, disease modeling, and preclinical drug testing.

Indolenine-Based Derivatives as Customizable Two-Photon Fluorescent Probes for pH Bioimaging in Living Cells.

Novel pH probes based on 2-(6-methoxynaphthalen-2-yl)-3,3-dimethyl-3H-indole have been synthesized and characterized. These compounds display excellent "off-on" fluorescence responses to acidic pH especially under two-photon (TP) excitation conditions as well as strong selectivity and sensitivity toward H+. These features are supported by fluorescence quantum yields over 35%, TP cross sections ∼60 GM, and good resistance to photodegradation under acidic conditions. The synthetic versatility of this model allows subcellular targets to be tuned through minor scaffold modifications without affecting its optical characteristics. The effectiveness of the probes' innate photophysical properties and the structural modifications for different pH-related applications are demonstrated in mouse embryonic fibroblast cells.

Development of the Myocardial Interstitium.

The space between cardiac myocytes is commonly referred-to as the cardiac interstitium (CI). The CI is a unique, complex and dynamic microenvironment in which multiple cell types, extracellular matrix molecules, and instructive signals interact to crucially support heart homeostasis and promote cardiac responses to normal and pathologic stimuli. Despite the biomedical and clinical relevance of the CI, its detailed cellular structure remains to be elucidated. In this review, we will dissect the organization of the cardiac interstitium by following its changing cellular and molecular composition from embryonic developmental stages to adulthood, providing a systematic analysis of the biological components of the CI. The main goal of this review is to contribute to our understanding of the CI roles in health and disease. Anat Rec, 302:58-68, 2019. © 2018 Wiley Periodicals, Inc.

Platinum-Doped Dendritic Structure as a Phosphorescent Label for Bacteria in Two-Photon Excitation Microscopy.

Herein, we present a water-soluble dendritric Pt(II) complex as a phosphorescent label for bacterial cells. The dendritic moiety endows the Pt(II) complex with unique properties such as water solubility, shielding from quenching by dioxygen, and binding to bacterial surfaces. The new biosensor was employed for two-photon excitation microscopy, and the binding was confirmed by electron microscopy, which demonstrates that such hybrid arrays can provide orthogonal yet complementary readouts.

Characteristics, applications and prospects of mesenchymal stem cells in cell therapy. / Características, aplicaciones y perspectivas de las células madre mesenquimales en terapia celular.

Recent advances in the field of cell therapy and regenerative medicine describe mesenchymal stem cells (MSCs) as potential biological products due to their ability to self-renew and differentiate. MSCs are multipotent adult cells with immunomodulatory and regenerative properties, and, given their therapeutic potential, they are being widely studied in order to evaluate their viability, safety and efficacy. In this review, we describe the main characteristics and cellular sources of MSCs, in addition to providing an overview of their properties and current clinical applications, as well offering updated information on the regulatory aspects that define them as somatic cell therapy products. Cell therapy based on MSCs is offered nowadays as a pharmacological alternative, although there are still challenges to be addressed in this regard.

In vitro self-assembly of proepicardial cell aggregates: an embryonic vasculogenic model for vascular tissue engineering.

Proepicardial/epicardial-derived cells are the main origin of the early embryonic coronary vascular bed. In vivo coronary vasculogenesis, which is a fast-occurring event, can be mimicked in vitro by culturing proepicardial tissue in different ways. The in vitro vasculogenic model presented in this study (a proepicardial suspension culture assay) partially reproduces coronary vascular development from its cellular precursors, a process known to be highly dependent on cell migration, cell differentiation, cell adhesion/sorting, and tissue fusion phenomena. The main aim of this study is to study the triggering signals and the cellular dynamics that regulate the differentiation of proepicardial cells into the angioblastic/endothelial lineage and their in vitro vasculogenic potential. Our results indicate that hanging drop-cultured proepicardia, which have an intrinsic vascular potential, behave like self-assembling cell aggregates or spheroids that can fuse to give rise to complex vascularized 3D structures. We believe that these self-assembling cell aggregates are an optimal choice to study the differentiation of coronary angioblasts, as well as a good method to reproduce vascular development in vitro. Finally, we propose the proepicardium as a suitable cellular source for vascular tissue engineering.

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro--correlation between contraction force and electrophysiology.

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential. We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.

In vivo and in vitro analysis of the vasculogenic potential of avian proepicardial and epicardial cells.

Coronary vessel formation is a special case in the context of embryonic vascular development. A major part of the coronary cellular precursors (endothelial, smooth muscle, and fibroblastic cells) derive from the proepicardium and the epicardium in what can be regarded as a late event of angioblastic and smooth muscle cell differentiation. Thus, coronary morphogenesis is dependent on the epithelial-mesenchymal transformation of the proepicardium and the epicardium. In this study, we present several novel observations about the process of coronary vasculogenesis in avian embryos, namely: (1) The proepicardium displays a high vasculogenic potential, both in vivo (as shown by heterotopic transplants) and in vitro, which is modulated by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor signals; (2) Proepicardial and epicardial cells co-express receptors for platelet-derived growth factor-BB and VEGF; (3) Coronary angioblasts (found all through the epicardial, subepicardial, and compact myocardial layers) express the Wilms' tumor associated transcription factor and the retinoic acid-synthesizing enzyme retinaldehyde-dehydrogenase-2, two markers of the coelomic epithelium involved in coronary endothelium development. All these results contribute to the development of our knowledge on the vascular potential of proepicardial/epicardial cells, the existent interrelationships between the differentiating coronary cell lineages, and the molecular mechanisms involved in the regulation of coronary morphogenesis.