Biomanufacturing of a Chitosan/Collagen Scaffold to Drive Adhesion and Alignment of Human Cardiomyocyte Derived from Stem Cells☆

Abstract The in vitro generation of a three-dimensional (3D) myocardial tissue employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration. Despite significant progresses in this field, cellular models are not yet able to provide a source of myocardial cells that will efficiently integrate and substitute damaged myocardial tissue. Stem cell-derived human cardiomyocytes (CMs) represent the most promising source for cardiac cell therapy. In order to sustain attachment, spreading, and orientation of human CMs on a scaffold we exploited an innovative negative replica patterning based on electrophoretic deposition to realize multi-scale micro-structured chitosan-collagen (C/C) scaffolds. Specific patterns were micro-structured on the cathode titanium disks using a laser machine. Cubic and hexagonal patterns were deeply characterized, and reproduced on the surface of the C/C scaffold. We initially challenged different blend with spontaneous contracting neonatal rat CMs to identificate the best substratum, finding that C/C 5:1 proportion can better sustain this type of culture. Finally, human CMs derived from induced pluripotent stem cells were seeded on these patterned scaffolds and colonization of the substrate was observed, thus confirming the validity of the chosen biomaterial. Moreover, preliminary experiments demonstrate the effectiveness of the pattern in controlling the orientation of human CMs. In conclusion, we designed and fabricated a scaffold that allows the attachment, spreading, and orientation of human CMs due to a correct C/C blend composition, to an innovative manufacturing process, and to an effective 3D architecture of the patterns. These data will surely help in solving the quest for a cardiac clinical patch

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

Using iPS Cells toward the Understanding of Parkinson's Disease

Cellular reprogramming of somatic cells to human pluripotent stem cells (iPSC) represents an efficient tool for in vitro modeling of human brain diseases and provides an innovative opportunity in the identification of new therapeutic drugs. Patient-specific iPSC can be differentiated into disease-relevant cell types, including neurons, carrying the genetic background of the donor and enabling de novo generation of human models of genetically complex disorders. Parkinson's disease (PD) is the second most common age-related progressive neurodegenerative disease, which is mainly characterized by nigrostriatal dopaminergic (DA) neuron degeneration and synaptic dysfunction. Recently, the generation of disease-specific iPSC from patients suffering from PD has unveiled a recapitulation of disease-related cell phenotypes, such as abnormal α-synuclein accumulation and alterations in autophagy machinery. The use of patient-specific iPSC has a remarkable potential to uncover novel insights of the disease pathogenesis, which in turn will open new avenues for clinical intervention. This review explores the current Parkinson's disease iPSC-based models highlighting their role in the discovery of new drugs, as well as discussing the most challenging limitations iPSC-models face today

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a PITX2 p.M200V mutation

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the specific PITX2 gene have been identified in patients with early-onset AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a PITX2 p.M200V mutation, using a commercially available non-integrating expression system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the PITX2 p.M200V mutation

Generation of induced Pluripotent Stem Cells as disease modelling of NLSDM

Neutral Lipid Storage Disease with Myopathy (NLSDM) is a rare defect of triacylglycerol metabolism, characterized by the abnormal storage of neutral lipid in organelles known as lipid droplets (LDs). The main clinical features are progressive myopathy and cardiomyopathy. The onset of NLSDM is caused by autosomal recessive mutations in the PNPLA2 gene, which encodes adipose triglyceride lipase (ATGL). Despite its name, this enzyme is present in a wide variety of cell types and catalyzes the first step in triacylglycerol lipolysis and the release of fatty acids. Here, we report the derivation of NLSDM-induced pluripotent stem cells (NLSDM-iPSCs) from fibroblasts of two patients carrying different PNPLA2 mutations. The first patient was homozygous for the c.541delAC, while the second was homozygous for the c.662G>C mutation in the PNPLA2 gene. We verified that the two types of NLSDM-iPSCs possessed properties of embryonic-like stem cells and could differentiate into the three germ layers in vitro. Immunofluorescence analysis revealed that iPSCs had an abnormal accumulation of triglycerides in LDs, the hallmark of NLSDM. Furthermore, NLSDM-iPSCs were deficient in long chain fatty acid lipolysis, when subjected to a pulse chase experiment with oleic acid. Collectively, these results demonstrate that NLSDM-iPSCs are a promising in vitro model to investigate disease mechanisms and screen drug compounds for NLSDM, a rare disease with few therapeutic options

Generation and characterization of the human iPSC line IDISi001-A isolated from blood cells of a CADASIL patient carrying a NOTCH3 mutation

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of hereditary stroke disorder. It is caused by mutations in NOTCH3 that lead to progressive degeneration of the smooth muscle cells in blood vessels. There is currently no treatment for this disorder. We reprogrammed to pluripotency blood mononuclear cells isolated from a patient carrying a NOTCH3 mutation by using a commercially available non-integrating system. The success in the generation of this iPSC line (IDISi001-A) suggests that the NOTCH3 mutation did not limit cell reprogramming and offers an unprecedented opportunity for studying and modeling CADASIL pathology

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a KCNA5 p.D322H mutation

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the atrial specific KCNA5 gene have been identified in patients with early-onset lone AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a KCNA5 p.D322H mutation, using a commercially available non-integrating system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the p.D322H mutation

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