Comparative analysis of mesenchymal stromal cells biological properties

The stromal progenitors of mesodermal cells, mesenchymal stromal cells (MSCs), are a heterogeneous population of plastic adherent fibroblast-like cells with extensive proliferative capacity and differentiation potential. Human MSCs have now been isolated from various tissues including bone marrow, muscle, skin, and adipose tissue, the latter being one of the most suitable cell sources for cell therapy, because of its easy accessibility, minimal morbidity, and abundance of cells. Bone marrow and subcutaneous or visceral adipose tissue samples were collected, digested with collagenase if needed, and seeded in Iscove's medium containing 5% human platelet lysate. Nonadherent cells were removed after 2-3 days and the medium was replaced twice a week. Confluent adherent cells were detached, expanded, and analyzed for several biological properties such as morphology, immunophenotype, growth rate, senescence, clonogenicity, differentiation capacity, immunosuppression, and secretion of angiogenic factors. The results show significant differences between lines derived from subcutaneous fat compared to those derived from visceral fat, such as the higher proliferation rate of the first and the strong induction of angiogenesis of the latter. We are convinced that the identification of the peculiarities of MSCs isolated from different tissues will lead to their more accurate use in cell therapy

Human iPSC modelling of a familial form of atrial fibrillation reveals a gain of function of I-f and I-CaL in patient-derived cardiomyocytes

Aims: Atrial fibrillation (AF) is the most common type of cardiac arrhythmias, whose incidence is likely to increase with the aging of the population. It is considered a progressive condition, frequently observed as a complication of other cardiovascular disorders. However, recent genetic studies revealed the presence of several mutations and variants linked to AF, findings that define AF as a multifactorial disease. Due to the complex genetics and paucity of models, molecular mechanisms underlying the initiation of AF are still poorly understood. Here we investigate the pathophysiological mechanisms of a familial form of AF, with particular attention to the identification of putative triggering cellular mechanisms, using patient's derived cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs). Methods and results: Here we report the clinical case of three siblings with untreatable persistent AF whose whole-exome sequence analysis revealed several mutated genes. To understand the pathophysiology of this multifactorial form of AF we generated three iPSC clones from two of these patients and differentiated these cells towards the cardiac lineage. Electrophysiological characterization of patient-derived CMs (AF-CMs) revealed that they have higher beating rates compared to control (CTRL)-CMs. The analysis showed an increased contribution of the If and ICaL currents. No differences were observed in the repolarizing current IKr and in the sarcoplasmic reticulum calcium handling. Paced AF-CMs presented significantly prolonged action potentials and, under stressful conditions, generated both delayed after-depolarizations of bigger amplitude and more ectopic beats than CTRL cells. Conclusions: Our results demonstrate that the common genetic background of the patients induces functional alterations of If and ICaL currents leading to a cardiac substrate more prone to develop arrhythmias under demanding conditions. To our knowledge this is the first report that, using patient-derived CMs differentiated from iPSC, suggests a plausible cellular mechanism underlying this complex familial form of AF

Generazione di cardiomiociti umani per lo studio di patologie cardiovascolari

Per capire i meccanismi molecolari che determinano una patologia, comprese le patologie cardiovascolari, il tipo di approccio biologico comunemente utilizzato consiste nel prelevare un frammento di tessuto interessato, isolare e coltivare le cellule da esso derivate e confrontare l’espressione genica e proteica di tali cellule con cellule “sane” di controllo. Tale approccio può essere applicato a patologie vascolari ma non a patologie cardiache, principalmente perché i cardiomiociti, a differenza delle cellule endoteliali o dei periciti, sono cellule talmente specializzate da aver perso ogni capacità replicativa, quindi molto difficili da mantenere in coltura per le analisi molecolari necessarie a determinare i processi patologici. E’ possibile tuttavia ottenere cardiomiociti umani a partire da cellule staminali. La ricerca del FRU lab si basa sulla recente scoperta del Dr. Yamanaka, che riguarda la creazione artificiale di cellule staminali indotte alla pluripotenza. Tali cellule si ottengono a partire da cellule della pelle, cellule del sangue o altri tipi cellulari il cui prelievo non comporta nessun danno al paziente, al massimo una piccola cicatrice. Per queste caratteristiche le cellule conservano il DNA del paziente, mantenendo quindi tutti i difetti genetici responsabili di patologie, comprese quelle cardiovascolari. Al momento ci stiamo occupando di capire le basi biologiche della fibrillazione atriale (FA), la patologia aritmica più diffusa nella popolazione al di sopra dei 60 anni di età. Nella maggior parte dei casi, FA è associata a fattori di rischio cardiaci come l’ipertensione, l’ischemia o malattie strutturali cardiache, tuttavia esiste un sottogruppo pari al 10-20 % del numero totale di pazienti che non presenta altri sintomi e rientra in una condizione chiamata “FA solitaria”. Una serie di studi ha dimostrato che FA e, in particolare “FA solitaria”, possono presentare una componente genetica. E’ stato recentemente dimostrato che il rischio individuale di sviluppare “FA solitaria” in giovane età aumenta drasticamente con l'aumento del numero di parenti con “FA solitaria” e che figli di genitori con FA hanno un rischio di sviluppo di FA circa raddoppiato. In collaborazione con i cardiochirurghi dell’Università di Brescia abbiamo identificato tre fratelli che hanno sofferto di una forma particolare di “FA solitaria” non rispondente a nessuna delle terapie utilizzate correntemente. Essendo una patologia comune ai tre fratelli abbiamo sospettato una forte componente genetica della patologia. Abbiamo chiesto a questi pazienti di donare un frammento di cute dal quale abbiamo isolato i fibroblasti che abbiamo riprogrammato, generando cellule staminali che dei pazienti mantengono lo stesso patrimonio genetico. Partendo da queste cellule staminali ottenute artificialmente abbiamo poi differenziato le cellule contrattili cardiache che hanno mantenuto il patrimonio genetico e quindi gli eventuali difetti, dei pazienti con FA. Il confronto di queste cellule con quelle derivate in parallelo da persone sane ci ha permesso di identificare alcuni fenomeni che potrebbero essere alla base della patologia come, ad esempio, una frequenza di pulsazione più elevata e una più alta soglia del potenziale d’azione

Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes

Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC

The Ferritin-Heavy-Polypeptide-Like-17 (FTHL17) gene encodes a ferritin with low stability and no ferroxidase activity and with a partial nuclear localization

Three functional ferritin genes have been identified so far in mammals, and they encode the cytosolic Heavy (FTH) and Light chain (FTL) and the mitochondrial ferritin. The expression of a transcript by a fourth ferritin-like gene (Ferritin-Heavy-Polypeptide-Like-17, FTHL17) on the X chromosome was reported in mouse spermatogonia and in early embryonic cells. METHODS: The intronless human FTHL17 gene encodes a protein with 64% identity to human FTH with substitution of key residues of the ferroxidase center. The gene was cloned into vectors for expression in Escherichia coli and mammalian cells, linked to a flag-tag. RESULTS: The recombinant FTHL17 from E. coli purified as an assembled 24-mer ferritin devoid of ferroxidase activity and with a reduced physical stability. When transiently expressed in mammalian cells the flag-FTHL17 assembled in ferritin shells that showed reduced stability to denaturants compared with flag H and L ferritins. Immunocytochemistry with anti-flag antibody decorated the nuclei of flag-FTHL17 transfected COS cells, but not those of the cells transfected with flag-FTH or flag-FTL. CONCLUSIONS: We concluded that FTHL17 encodes a ferritin-like protein without ferroxidase activity. Its restricted embryonic expression and partial nuclear localization suggest that this novel ferritin type may have functions other than iron storage. GENERAL SIGNIFICANCE: The work confirms the presence of a fourth functional human ferritin gene with properties distinct from the canonical cytosolic ones