Vai ai contenuti. | Spostati sulla navigazione | Spostati sulla ricerca | Vai al menu | Contatti | Accessibilità

| Crea un account

Contato, Anna (2016) Cardiomyocytes generation by programming human pluripotent stem cell fate in microfluidics: from Wnt pathway modulators to synthetic modified mRNA. [Tesi di dottorato]

Full text disponibile come:

[img]Documento PDF (Tesi di Dottorato)
Tesi non accessible fino a 31 Luglio 2019 per motivi correlati alla proprietà intellettuale.
Visibile a: nessuno


Abstract (inglese)

Cardiovascular disease (CVD) is still one of the major cause of morbidity and mortality in the world, with ischemic heart disease representing the majority of deaths over the past 10 years. The high burden of the disease, both immediate and chronic, associated with the high costs for the healthcare systems, claim for the development of novel therapeutic strategies. The main issue of current pharmacological and interventional therapeutic approaches is their inability to compensate the great and irreversible loss of functional cardiomycytes (CMs). Because of the limited regenerative capacity of post-natal CMs and the difficulty to obtain and isolate heart bioptic tissue, very limited supplies of these cells are available at present for dedicated studies. Moreover, even if animal models are surely the best tool to study and understand in vivo the mechanisms of specific human pathologies in a complex organism, they are not fully predictive and representative of the human condition; from an economic point of view, animal maintenance and the related experimentations are time consuming and very expensive.
In this scenario, human pluripotent stem cells (hPSCs), including human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs), play an important role in the cardiovascular research field, because they can be indefinitely expanded in culture without loosing their stemness, and differentiated into cells of the three germ layers, such as CMs. A great breakthrough in science has occurred in 2007 with the discovery of hiPSCs by the Nobel Prize Shinya Yamanaka. This has been the starting point for deriving patient-specific hiPSCs from the reprogramming of somatic cells obtained with less- or non-invasive procedures (skin biopsies, blood, urine…), useful for the generation of tissues for autologous-repair, bypassing the ethical and political debates surrounding the hESCs derivation.
The researchers have made several efforts to develop strategies to efficiently direct hPSCs cardiac differentiation and the existing methods for deriving CMs involve stage-specific perturbations of different signaling pathways using growth factors (GFs) or small molecules that recapitulate key steps of the cardiac development observed in vivo. However, these strategies are accompanied by some limitations including: high intra- and inter-experimental variability, low efficiencies, presence of xeno-contaminants, undefined medium components and differences in the expression of cytokines of endogenous signaling pathways. Other strategies are based on the direct lineage conversion of somatic cells, especially fibroblasts, via the overexpression of cardiac transcription factors (TFs) combinations through integrating and non-integrating vectors. However, also these approaches are characterized by low efficiencies, combined with the risk of genomic integration and insertional mutagenesis when using integrating vectors or the need for stringent steps of purification when using non-integrating techniques. Because of the difficulty to specifically direct hPSCs cardiac fate in a robust way, combined with the scarce ability of conventional culture systems to reproduce in vitro, the environment in which cells reside in vivo, the CMs produced to date are immature and more similar to fetal cardiac cells.
In 2010, Warren L. and co-workers pioneered a novel, non-integrating strategy based on repeated transfection with cathionic vehicles of synthetic modified messenger RNA (mmRNA), specifically designed to avoid innate immune response from the cell, demonstrating the possibility to both reprogram somatic cells to pluripotency and to programm hPSCs fate into terminally differentiated myogenic cells.
Hence, the aim of this PhD thesis is the development of an efficient and robust method for cardiac differentiation of hPSCs by combining the mmRNA with the microfluidic technology. Repeated transfections with mmRNA encoding 6 cardiac TFs are employed to force the endogenous protein expression in the cells and to drive the differentiation toward functional maturation of CMs. The integration of cardiac differentiation within an ad hoc microfluidic platform, facbricated in BioERA laboratory, allows a more precise control of culture conditions, enabling a high mmRNA transfection efficiency, thanks to the high volume/surface ratio, and the in vitro reproduction of physiological niches. In fact, the small scale offered by microfluidics, best mimics the cellular dynamics, which occur in the soluble microenvironment in vivo. Moreover, the microfluidic technology offers the possibility to perform combinatorial, multiparametric, parallelized and highthroughput experiments at one time in a cost-effective manner, not achievable and not economically sustainable in macroscopic conventional culture systems.
Chapter 1 starts with the definition of regenerative medicine and introduces the complexity of cardiac development, with the network of TFs that cooperate in this process. The state of the art regarding the derivation of CMs from hPSCs and from the transdifferentiation of somatic cells is described, together with the current limitations and challenges. Finally, the general aim of this PhD thesis is presented.
Chapter 2 will focus on hPSCs (hES and hiPS) employed during this project, describing their most important characteristics. It will be also presented a monolayer-based cardiac differentiation protocol of hPSCs that, to date is considered the gold standard for the fast generation of a high yield of beating CMs in conventional culture systems. This protocol relies on the temporal modulation of Wnt pathway via the administration of small molecules. In addition, a hES line, dual reporter for 2 cardiac TFs will be described and always adopted as a tool to monitor the progression of cardiac differentiation. The results obtained in standard cultures will be showed.
Chapter 3 will review the state of the art of microfluidic technology for cell culture in regenerative medicine applications. Then, the microfluidic platform fabrication will be described and employed, followed by the optimization of culture, expansion and cardiac differentiation of hPSCs with the gold standard protocol deriving form the translation from macro- to micro-scale.
Chapter 4 will introduce the novel mmRNA strategy for reprogramming and programming cell fate: also in this case the state of the art will be discussed. Then, the experimental strategies developed to program cardiac differentiation of hPSCs toward a more mature CM phenotype will be presented, together with the results obtained and the related structural, functional and molecular characterizations. In this work, for the first time, it has been possible to derive CMs from hPSCs with repeated transfections of mmRNA encoding 6 cardiac TFs in microfluidics, with efficiencies higher to current methods described in literature, performed in standard systems.
Finally, Chapter 5 will present the general discussion and conclusions, with the future perspectives regarding the use of mmRNA combined with microfluidic technology for deriving different CMs phenotypes, just varying the combination of TFs delivered.
To conclude, the experiments developed during this project provide proof-of-principle that it is possible to program hPSCs fate toward cardiac lineage and cardiac maturation in microfluidics; moreover, thanks to the non-integrating characteristic of mmRNA, the CMs obtained are clinical-grade and could potentially be employed in the next future for clinical applications of autologous tissue self-repair and for personalized drug screening.

Abstract (italiano)

Le malattie cardiovascolari rappresentano ad oggi una delle principali cause di morbidità e mortalità nel mondo, tra le quali la patologia ischemica è responsable del maggior numero di decessi negli ultimi 10 anni. L’elevato impatto determinato da tali patologie, sia acute che croniche, e gli elevati costi per i sistemi sanitari, richiedono lo sviluppo di nuove strategie terapeutiche.
La questione principale riguardante gli attuali approcci terapeutici, sia farmacologici sia interventistici, è rappresentata dalla loro incapacità di compensare l’elevata ed irreversibile perdita di cardiomiociti funzionali. A causa della limitata capacità rigenerativa dei cardiomiociti post-natali e della difficoltà di reperire ed isolare tessuto cardiaco bioptico, scarse sono le fonti di tali cellule disponibili per uno studio dedicato. Tra l’altro, anche se i modelli animali ancora oggi rappresentano sicuramente lo stumento migliore per studiare e comprendere in vivo i meccanismi alla base dello sviluppo di specifiche patologie umane, nel constesto di un organismo complesso, essi non sono completamente predittivi e rappresentativi della condizione umana analizzata; da un punto di vista economico, il mantenimento di tali animali e le relative sperimentazioni, richiedono molto tempo e costi elevati.
In questo scenario, le cellule staminali umane pluripotenti (hPSCs), comprese le cellule staminali embrionali (hESCs) e le cellule staminali pluripotenti indotte (hiPSCs), rivestono un ruolo importante nella ricerca cardiovascolare perché possono essere espanse in coltura indefinitamente, senza perdere la loro staminalità, e differenziare nelle cellule che componogono i tre foglietti germinativi, come ad esempio i cardiomiociti. Un’importante svolta nella ricerca scientifica è avvenuta nel 2007, con la scoperta delle hiPSCs da parte del Premio Nobel Shinya Yamanaka. Ciò ha rappresentato il punto di partenza per derivare hiPSCs paziente-specifiche attraverso il reprogramming di cellule somatiche ottenute con procedure mini- o non-invasive (derivate da biopsie cutanee, sangue, urina…), utili per generare tessuti per una riparazione autologa, evitando i problemi etici e politici relativi alla derivazione delle hESCs. Notevoli studi sono stati condotti dai ricercatori nel tentativo di sviluppare strategie che efficientemente ed in maniera robusta guidino il differenziamento cardiaco delle hPSCs, basate sulla perturbazione stadio-specifica di differenti vie di segnalazione, mediante l’uso di fattori di crescita e piccole molecole, che ricapitolano i punti essenziali dello sviluppo cardiaco osservato in vivo. Tuttavia, questi metodi sono accompagnati da alcune limitazioni, quali: elevata variabilità intra ed inter-sperimentale, presenza di xeno-contaminanti, componenti indefinite nei medium di coltura e differenze nei livelli di espressione di citochine endogene. Altre strategie si basano invece sulla conversione diretta di cellule somatiche, specialmente fibroblasti, attraverso l’overespressione di una combinazione di fattori di trascrizione cardiaci mediante vettori integrativi e non-integrativi; tuttavia, anche tali approcci sono caratterizzati da basse efficienze nella generazione di cardiomiociti, associate al rischio di integrazioni genomiche e mutagenesi inserzionale nel caso dei vettori integrativi, o alla necessità di effettuare diversi step di purificazione quando si ultilizzano sistemi non-integrativi. Pertanto, a causa delle difficoltà dei sistemi convenzionali di coltura nel dirigere specificamente ed in maniera robusta il differenziamento cardiaco delle hPSCs, assieme alla scarsa capacità di riprodurre in vitro l’ambiente in cui le cellule risiedono in vivo, i cardiomiociti prodotti attualmente sono immaturi e più simili allo stadio fetale di sviluppo.
Nel 2010 Warren L. ed il suo gruppo di ricerca ha sperimentato per la prima volta una tecnologia innovativa di tipo non-integrativo basata su trasfezioni ripetute con lipidi cationici di RNA messaggeri modificati sinteticamente (mmRNA) per evitare la risposta immunitaria innata da parte delle cellule; egli ha dimostrato la possibilità sia di riprogrammare cellule somatiche allo stato pluripotente, sia di programmare il differenziamento miogenico di hiPSCs.
Pertanto, lo scopo di questa tesi di dottorato è quello di sviluppare un metodo robusto ed efficiente per il differenziamento cardiaco di hPSCs combinando gli mmRNA con la tecnologia microfluidica. Ripetute trasfezioni di mmRNA codificanti per 6 fattori di trascrizione coinvolti nello sviluppo e nel funzionamento cardiaco, vengono impiegate per forzare l’espressione proteica endogena delle cellule e per guidare il differenziamento verso la maturazione funzionale dei cardiomiociti. L’integrazione del differenziamento cardiaco in una piattaforma microfluidica ad hoc, prodotta nel laboratorio BioERA, consente un controllo più preciso delle condizioni di coltura garantendo un’elevata efficienza di trasfezione degli mmRNA grazie all’elevato rapporto superficie/volume e permette la riproduzione in vitro di nicchie fisiologiche. Infatti, la miniaturizzazione consente di mimare al meglio le dinamiche cellulari che avvengono in vivo nel microambiente solubile. Le tecnologia microfluidica offre la possibilità di effettuare esperimenti combinati, multiparametrici e paralleli in una sola volta e con elevato rendimento a costi ridotti, non realizzabili nei macroscopici e costosi sistemi di coltura convenzionali.
Il Capitolo 1 inizia con la definizione di medicina rigenerativa e introduce la complessità dello sviluppo cardiaco ed il network di fattori di trascrizione che cooperano durante questo processo. Viene poi descritto lo stato dell’arte relativo alle strategie per l’ottenimento di cardiomiociti da hPSCs e al transdifferenziamento cardiaco di cellule somatiche, insieme alle relative limitazioni e alle problematiche attuali da risolvere. Infine viene presentato lo scopo generale di questa tesi di dottorato.
Il Capitolo 2 si focalizzerà sulle hPSCs (sia hES sia hiPS) impiegate durante questo progetto, descrivendo le caratterisatiche principali di tali cellule. Verrà inoltre presentato un protocollo di differenziamento cardiaco di hPSCs in monostrato che attualmente è considerato il gold standard per ottenere velocemente un’elevata resa di cardiomiociti contrattili in supporti di coltura convenzionali. Tale protocollo si basa sulla modulazione del pathway canonico di Wnt attraverso l’applicazione di due piccole molecole. Inoltre, una linea di hES, doppio reporter per 2 fattori di trascrizione cardiaci, verrà descritta ed impiegata in tutti gli esperimenti come strumento per monitorare l’andamento del differenziamento cardiaco delle hPSC. I risultati ottenuti in colture standard verranno mostrati.
Il Capitolo 3 esaminerà lo stato dell’arte della tecnologia microfluidica nelle applicazioni di medicina rigenerativa, sottolineando i vantaggi derivanti dalla combinazione della microtecnologia con la biologia cellulare. Verrà successivamente descritta la fabbricazione della piattaforma microfluidica utilizzata, con la successiva ottimizzazione della coltura, espansione e differenziamento cardiaco gold standard delle hPSCs conseguenti alla conversione dalla macro- alla microscala.
Il Capitolo 4 introdurrà la nuova strategia degli mmRNA per la riprogrammazione e la programmazione cellulare: anche in tal caso verrà discusso lo stato dell’arte. In seguito, verranno presentate le strategie sperimentali sviluppate per programmare il differenziamento cardiaco delle hPSCs verso un fenotipo più maturo dei cardiomiociti, insieme ai risultati ottenuti con le relative caratterizzazioni strutturali, funzionali e molecolari. In questo lavoro, per la prima volta, è stato possibile ottenere cardiomiociti da hPSCs attraverso ripetute trasfezioni di mmRNA per 6 fattori di trascrizione cardiaci in microfluidica, con efficienze superiori rispetto ai metodi presenti attualmente in letteratura, svolti in sistemi convenzionali.
Il Capitolo 5 infine presenterà la discussione e le conclusioni generali, assieme alle prospettive future riguardanti l’uso degli mmRNA combinati con la microfluidica per ottenere diversi fenotipi di cardiomiociti, variando la combinazione di fattori di trascrizione veicolati. In conclusione, gli esperimenti sviluppati in questo progetto di dottorato forniscono un proof-of-principle della possibilità di programmare con gli mmRNA il destino delle hPSCs verso il differenziamento e la maturazione di cardiomiociti funzionali in microfluidica; inoltre, essendo gli mmRNA una strategia non-integrativa , i cardiomiociti ottenuti in questo modo possono essere impiegati nel prossimo futuro per applicazioni cliniche di ricostruzione tissutale autologa e per screening farmacologici personalizzati.

Tipo di EPrint:Tesi di dottorato
Relatore:Piccolo, Stefano
Correlatore:Elvassore, Nicola
Dottorato (corsi e scuole):Ciclo 28 > Scuole 28 > BIOMEDICINA > MEDICINA RIGENERATIVA
Data di deposito della tesi:27 Luglio 2016
Anno di Pubblicazione:31 Luglio 2016
Parole chiave (italiano / inglese):Human Pluripotent Stem Cells, Cardiomyocytes, Small molecules, signaling pathway, microfluidics, synthetic modified mRNA, mmRNA
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Molecolare
Codice ID:9694
Depositato il:03 Nov 2017 10:22
Simple Metadata
Full Metadata
EndNote Format


I riferimenti della bibliografia possono essere cercati con Cerca la citazione di AIRE, copiando il titolo dell'articolo (o del libro) e la rivista (se presente) nei campi appositi di "Cerca la Citazione di AIRE".
Le url contenute in alcuni riferimenti sono raggiungibili cliccando sul link alla fine della citazione (Vai!) e tramite Google (Ricerca con Google). Il risultato dipende dalla formattazione della citazione.

[1]. Appasani K, Appasani R, K. 2011. Stem Cells & Regenerative Medicine From Molecular Embriology to Tissue Engineering. London: Humana Press. Cerca con Google

[2]. Laflamme MA, Murry CE. 2005. Regenerating the heart. Nature Biotechnology 23:845-856. Cerca con Google

[3]. Whitesides GM. 2006. The origins and the future of microfluidics. Nature 442:368-373. Cerca con Google

[4]. Harink B, Le Gac S, Truckenmu R, van Bitterswijka C, Habibovic P. 2013. Regeneration-on-a-chip? The perspective on use of microfluidics in regenerative medicine. Lab on a Chip 13:3512-3528. Cerca con Google

[5]. Lewis T. 2016. Human Heart: Anatomy, Function & Facts. Livescience website. www.livescience. com. Vai! Cerca con Google

[6]. Patton KT, Thibodeau, GA. 1996. Anatomy and Physiology. 8th Edition: MOSBY Elsevier. Cerca con Google

[7]. Olivetti G, Cigola E, Maestri R, Corradi D, Lagrasta C, Gambert SR, Anversa P. 1996. Aging, Cardiac Hypertrophy and Ischemic Cardiomyopathy Do Not Affect the Proportion of Mononucleated and Multinucleated Myocytes in the Human Heart, Journal of Molecular and Cellular Cardiology. Journal of Molecular and Cellular Cardiology 28:1463-1477. Cerca con Google

[8]. Gerecht-Nir S, Radisic M, Park H, Cannizzaro C, Boublik J, Langer R. 2006. Biophysical regulation during cardiac development and application to tissue engineering. The Internal Journal of Developmental Biology 50(2-3):233-243. Cerca con Google

[9]. Bird SD, Doevendans PA, van Rooijen MA, de la Riviere AB, Hassink RJ, Passier R, Mummery CL. 2003. The human adult cardiomyocyte phenotype. Cardiovascular Research 58:423-434. Cerca con Google

[10]. Carol CG, Parker B, Antin PB. 2000. To the heart of myofibril assembly. Trends in Cell Biology 10:355-362. Cerca con Google

[11]. Vai! Cerca con Google

[12]. Cheng H, Lederer WJ, Cannell MB. 1993. Calcium sparks: elementary events underlying exitation-contraction coupling in heart muscle. Science 262:740-744. Cerca con Google

[13]. Cannell MB, Cheng H, Lederer WJ. 1994. Spatial non-uniformities in [Ca2+]i during exitation-contraction coupling in cardiac myocytes. Biophysical Journal 67:1942-1956. Cerca con Google

[14]. Crespo LM, Grantham CJ, Cannell MB. 1990. Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes. Nature 345(6276):618-621. Cerca con Google

[15]. Austin Community College District website, Vai! Cerca con Google

[16]. Feric NT, Radisic M. 2016. Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues. Adv Drug Deliv Rev 96:110-134. Cerca con Google

[17]. Porter GA, Hom J, Hoffman D, Quintanilla R, de Mesy Bentley K, Sheu SS. 2011. Bioenergetics, mitochondria, and cardiac myocyte differentiation. Progress in Pediatric Cardiology 31:75-81. Cerca con Google

[18]. Navaratnam V. 1987. Heart Muscle: Ultrastractural Studies. Cambridge University Press, New York. Cerca con Google

[19]. Mc Culley DJ, Black BL. 2012. Transcription Factor Pathways and Congenital Heart Disease. Current topics in developmental biology. 100:253-277. Cerca con Google

[20]. Srivastava D. 2006. Genetic regulation of cardiogenesis and congenital heart disease. Annual Review of Pathology: Mechanisms of Disease 1:199-213. Cerca con Google

[21]. Burridge P, Keller G, Gold JD, Wu JC. 2012. Production of de novo cardiomyocytes: human pluripotent stem cells differentiation and direct reprogramming. Cell Stem Cell 10:16-28. Cerca con Google

[22]. Buckingham M, Mailhac S, Zaffran S. 2005. Building the mammalian heart from two sources of myocardial cells. Nature Reviews Genetics 6:826-835. Cerca con Google

[23]. Kwon C, Arnold J, Hsiao EC, Taketo MM, Conklin BR, Srivastava D. 2007. Canonical Wnt signaling is a positive regulator of mammalian cardiac progenitors. Proceedings of the National Academy of Sciences 104:10894-10899. Cerca con Google

[24]. Person AD, Klewer SE, Runyan RB. 2005. Cell Biology of Cardiac Cushion Development. Int Rev Cytol 243:287-335. Cerca con Google

[25]. Evans SM, Yelon D, Conlon FL, Kirby ML. 2010. Myocardial Lineage Development. Circulation research 107:1428-1444. Cerca con Google

[26]. Arnold SJ, Robertson EJ. 2009. Making a committment: cell lineage allocation and axis patterning in the early mouse embryo. Nature Reviews Molecular Cell Biology 10:91-103. Cerca con Google

[27]. Später D, Hansson EM, Zangi L, Chien KR. 2014. How to make a cardiomyocyte. Development 141:4418-4431. Cerca con Google

[28]. Noseda M, Peterkin T, Simões FC, Patient R, Schneider MD. 2011. Cardiopoietic Factors: Extracellular Signals for Cardiac Lineage Commitment. Circulation Research 108:129-152. Cerca con Google

[29]. Bondue A, Blanpain C. 2010. Mesp1: A Key Regulator of Cardiovascular Lineage Commitment. Circulation Research 107:1414-1427. Cerca con Google

[30]. Costello I, Pimeisl IM, Drager S, Bikoff EK, Robertson EJ, Arnold SJ. 2011. The T-box transcription factor Eomesodermin acts upstream of Mesp1 to specify cardiac mesoderm during mouse gastrulation. Nature Cell Biology 13:1084-1091. Cerca con Google

[31]. David R, Brenner C, Stieber J, Schwarz F, Brunner S, Vollmer M, Mentele E, Muller-Hocker J, Kitajima S, Lickert H. 2008. Mesp1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling. Nature Cell Biology 10:338-345. Cerca con Google

[32]. Naito AT, Shiojima I, Akazawa H, Hidaka K, Norisaki T, Kikuchi A, Komuro I. 2006. Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and haematopoiesis. Proceedings of the National Academy of Sciences 103:19812-19817. Cerca con Google

[33]. Ueno S, Weidinger G, Osugi T, Kohn AD, Golob JL, Pabon L, Reinecke H, Moon RT, Murry CE. 2007. Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proceedings of the National Academy of Sciences 2007:9685-9690. Cerca con Google

[34]. Kwon C, Qian L, Cheng P, Nigam V, Arnold J, Srivastava D. 2009. A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitors. Nature Cell Biology 11:951-957. Cerca con Google

[35]. Qyang Y, Martin-Puig S, Chiravuri M, Chen S, Xu H, Bu L, Jiang X, Lin L, Granger A, Moretti A. 2007. The renewal and differentiation of Isl+ cardiovascular progenitors are controlled by Wnt/beta-catenin pathway. Cell stem cell 1:165-179. Cerca con Google

[36]. Sahara M, Santoro F, Chien KR. 2015. Programming and reprogramming a human heart cell. The EMBO Journal 34:710-738. Cerca con Google

[37]. Cannon B. 2013. Cardiovascular disease: Biochemistry to behaviour. Nature 2-3. Cerca con Google

[38]. Laflamme MA, Murry CE. 2011. Heart regeneration. Nature 473:326-335. Cerca con Google

[39]. Jiang J, Han P, Zhang Q, Zhao J, Ma Y. 2012. Cardiac differentiation of human pluripotent stem cells. J Cell Mol Med 16:1663-1668. Cerca con Google

[40]. Fiedler LR, Maifoshie E, Schneider MD. 2014. Chapter Four - Mouse Models of Heart Failure: Cell Signaling and Cell Survival. Curr Top Dev Biol 109:171-247. Cerca con Google

[41]. Olson H, Betton G, Robinson D. 2000. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regulatoty Toxicology and Pharmacology 32:56-67. Cerca con Google

[42]. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. 1998. Embryonic Stem Cell Lines Derived From Human Blostocysts. Science 282:1145-1147. Cerca con Google

[43]. Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells from mouse embryonic and adut fibroblast cultures by defined factors. Cell 126:663-676. Cerca con Google

[44]. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined culture. Cell 131:861-872. Cerca con Google

[45]. Ebert AD, Diecke S, Chen IJ, Wu JC. 2015. Reprogramming and transdifferentiation for cardiovascular development and regenerative medicine: where do we stand? EMBO Molecular Medicine 7(9):1090-1103. Cerca con Google

[46]. Keller G. 2005. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes & Development 19:1129-1155. Cerca con Google

[47]. Nam Y, Song K, Luo X, Daniel E, Lambeth K, West K, Hill JA, DiMaio JM, Baker LA, Bassel-Duby R, Olson EN. 2013. Reprogramming of human fibroblasts toward a cardiac fate. Proceedings of the National Academy of Sciences 110:5588-5593. Cerca con Google

[48]. Ieda M, Fu J, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D. 2010. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell 142:375-386. Cerca con Google

[49]. Warren L, Manos PD, Ahfeldt T, Loh Y, Li H, Lau F, Ebina W, Mandal P, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ. 2010. Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA. Cell stem cell 7:618-630. Cerca con Google

[50]. Warren L, Ni Y, Wang J, Guo X. 2012. Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA. Nature. 2(657):1-7. Cerca con Google

[51]. Chang CW, Lay YS, Pawlik KM, Liu K, Sun CW, Li C, Schoeb TR, Townes TM. 2009. Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells 27:1042-1049. Cerca con Google

[52]. Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. 2009. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85:348-362. Cerca con Google

[53]. Davis RP, van den Berg CW, Casini S, Braam SR, Mummery CL. 2011. Pluripotent stem cell models of cardiac disease and their implication for drug discovery and development. Trends Mol Med 17:475-484. Cerca con Google

[54]. Mummery C, Ward-van Oostwaard D, Doevendans P, Spijker R, van den Brink S, Hassink R, van der Heyden M, Opthof T, Pera M, de la Riviere AB, Passier R, Tertoolen L. 2003. Differentiation of Human Embryonic Stem Cells to Cardiomyocytes: Role of Coculture With Visceral Endoderm-Like Cells. Circulation 107:2733-2740. Cerca con Google

[55]. Filipczyc AA, Passier R, Rochat A, Mummery CL. 2007. Cardiovascular development: towards biomedical applicability. Cellular and Molecular Life Sciences 64(6):704-718. Cerca con Google

[56]. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Gepstein L. 2001. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. Journal of Clinical Investigation 108:407-414. Cerca con Google

[57]. Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A, Ellis J, Keller GM. 2011. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell stem cell 8:228-240. Cerca con Google

[58]. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM. 2008. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453:524-528. Cerca con Google

[59]. Cai W, Albini S, Wei K, Willems E, Guzzo RM, Tsuda M, Giordani L, Spiering S, Kurian L, Yeo GW, Puri PL, Mercola M. 2013. Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment. Genes & Development 27:2332-2344. Cerca con Google

[60]. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, Reinecke H, Xu C, Hassanipour M, Police S. 2007. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nature Biotechnology 25:1015-1024. Cerca con Google

[61]. Mummery CL, Zhang J, Ng ES, Elliott DA, Elefanty AG, Kamp TJ. 2012. Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells to Cardiomyocytes: A Methods Overview. Circulation Research 111:344-358. Cerca con Google

[62]. Paige SL, Osugi T, Afanasiev OK, Pabon L, Reinecke H, Murry CE. 2010. Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells. PLoS ONE 5:1. Cerca con Google

[63]. Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, Raval KK, Zhang J, Kamp TJ, Palecek SP. 2012. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proceedings of the National Academy of Sciences 109:E1848-E1857. Cerca con Google

[64]. Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine L, Bao X, Hsiao C, Kamp TJ, Palecek SP. 2013. Directed cardiomyocytes differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nature Protocols 8:162-175. Cerca con Google

[65]. Xu H, Yi BA, Wu H, Bock C, Gu H, Lui KO, Park J-C, Shao Y, Riley AK, Domian IJ. 2012. Highly efficient derivation of ventricular cardiomyocytes from induced pluripotent stem cells with a distinct epigenetic signature. Cell Research 22:142-154. Cerca con Google

[66]. Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, Lan F, Diecke S, Huber B, Mordwinkin NM. 2014. Chemically defined generation of human cardiomyocytes. Nature methods 11:855-860. Cerca con Google

[67]. Keung W, Boheler K, Li R. 2014. Developmental cues for the maturation of metabolic, electrophysiological and calcium handling properties of human pluripotent stem cell-derived cardiomyocytes. Stem Cell Research & Therapy 5:17. Cerca con Google

[68]. Lundy SD, Zhu WZ, Regnier M, Laflamme MA. 2013. Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells. Stem Cells and Development 22:1991-2002. Cerca con Google

[69]. Robertson C, Tran DD, George SC. 2013. Concise review: maturation phases of human pluripotent stem cell-derived cardiomyocytes. Stem Cells 31:829-837. Cerca con Google

[70]. Piquereau J, Caffin F, Novotova M, Lemaire C, Veksler V, Garnier A, Ventura-Clapier R, Joubert F. 2013. Mitochondrial dynamics in the adult cardiomyocytes: which roles for a highly specialized cell? Frontiers in Physiology Journal 4:102. Cerca con Google

[71]. Moore JC, Fu J, Chan YC, Lin D, Tran H, Tse HF, Li RA. 2008. Distinct cardiogenic preferences of two human embryonic stem cell (hESC) lines are imprinted in their proteomes in the pluripotent state. Biochemical and Biophysical Research Communications 372:553-558. Cerca con Google

[72]. Cao F, Wagner RA, Wilson KD, Xie X, Fu JD, Drukker M, Lee A, Li RA, Gambhir SS, Weissman IL, Robbins RC, Wu JC. 2008. Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes. PLoS ONE 3:e3474. Cerca con Google

[73]. Ma J, Guo L, Fiene SJ, Anson BD, Thomson JA, Kamp TJ, Kolaja KL, Swanson BJ, January CT. 2011. High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents. American Journal of Physiology - Heart and Circulatory Physiology 301:2006-2017. Cerca con Google

[74]. Yanagi K, Takano M, Narazaki G, Uosaki H, Hoshino T, Ishii T, Misaki T, Yamashita JK. 2007. Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels and T-Type Calcium Channels Confer Automaticity of Embryonic Stem Cell-Derived Cardiomyocytes. Stem Cells 25:2712-2719. Cerca con Google

[75]. Zahanich I, Sirenko SG, Maltseva LA, Tarasova YS, Spurgeon HA, Boheler KR, Stern MD, Lakatta EG, Maltsev VA. 2011. Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling. J Mol Cell Cardiol 50:66-76. Cerca con Google

[76]. Caspi O, Itzhaki I, Kehat I, Gepstein A, Arbel G, Huber I, Satin J, Gepstein L. 2009. In vitro electrophysiological drug testing using human embryonic stem cell derived cardiomyocytes. Stem Cells and Development 18:161-172. Cerca con Google

[77]. Kadir SHSA, Ali NN, Mioulane M, Brito-Martins M, Abu-Hayyeh S, Foldes G, Moshkov AV, Williamson C, Harding SE, Gorelik J. 2009. Embryonic stem cell-derived cardiomyocytes as a model to study fetal arrhythmia related to maternal disease. J Cell Mol Med 13:3730-3741. Cerca con Google

[78]. Xu XQ, Soo SY, Sun W, Zweigerdt R. 2009. Global expression profile of highly enriched cardiomyocytes derived from human embryonic stem cells. Stem Cells 27:2163-2174. Cerca con Google

[79]. Synnergren J, Akesson K, Dahlenborg K, Vidarsson H, Ameen C, Steel D, Lindahl A, Olsson B, Sartipy P. 2008. Molecular signature of cardiomyocyte clusters derived from human embryonic stem cells. Stem Cells 26:1831-1840. Cerca con Google

[80]. Yigang W. 2014. Myocardial Reprogramming Medicine: The Development, Application, and Challenge of Induced Pluripotent Stem Cells. New Journal of Science 2014:1-22. Cerca con Google

[81]. Ban K, Wile B, Kim S. 2013. Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA. Circulation 128:1897-1909. Cerca con Google

[82]. Potten CS, Loeffler M. 1990. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 110:1001-1020. Cerca con Google

[83]. Wert Gd, Mummery C. 2003. Human embryonic stem cells: research, ethics and policy. Human Reproduction 18:672-682. Cerca con Google

[84]. Mitalipov S, Wolf D. 2009. Totipotency, pluripotency and nuclear reprogramming. Advances in Biochemical Engineering & Biotechnology 114:185-199. Cerca con Google

[85]. Berdasco M, Esteller M. 2011. DNA methylation in stem cell renewal and multipotency. Stem Cell Research & Therapy 2:1-9. Cerca con Google

[86]. Steinhoff G. 2011. Regenerative Medicine - from Protocol to Patient. Journal of Stem Cells & Regenerative Medicine 7:57-58. Cerca con Google

[87]. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T. 2001. Neural progenitors from human embryonic stem cells. Nature Biotechnology 19:1134-1140. Cerca con Google

[88]. Narsinh KH, Plews J, Wu JC. 2011. Comparison of Human Induced Pluripotent and Embryonic Stem Cells: Fraternal or Identical Twins? Molecular Therapy 19:635-638. Cerca con Google

[89]. Baker M. 2007. Adult cells reprogrammed to pluripotency without tumors. Nature Reports Stem Cells 124:1038. Cerca con Google

[90]. Yu J, Vodyanic MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. 2007. Induced Pluripotent Stem Cell Lines derived from Human Somatic Cells. Science 318(5858):1917-1920. Cerca con Google

[91]. Zhou T, Beda C, Dunzinger S, Huang Y, Ho JC, Yang J, Wang Y, Zhang Y, Zhuang Q, Li Y, Bao X, Tse H, Grillari J, Grillari-Voglauer R, Pei D, Esteban MA. 2012. Generation of human induced pluripotent stem cells from urine samples. Nature Protocols 7:2080-2089. Cerca con Google

[92]. Den Hartogh SC, Schreurs C, Monshouwer-Kloots JJ, Davis RP, Elliott DA, Mummery CL, Passier R. 2015. Dual Reporter MESP1mCherry/w-NKX2-5eGFP/w hESCs Enable Studying Early Human Cardiac Differentiation. Stem Cells 33:56-67. Cerca con Google

[93]. Den Hartogh SC, Passier R. 2016. Concise Review: Fluorescent Reporters in Human Pluripotent Stem Cells: Contributions to Cardiac Differentiation and Their Applications in Cardiac Disease and Toxicity. Stem Cells 34:13-26. Cerca con Google

[94]. Giobbe GG, Michielin F, Luni C, Giulitti S, Martewicz S, Dupont S, Floreani A, Elvassore N. 2015. Functional differentiation of human pluripotent stem cells on a chip. Nature Methods 12:637-640. Cerca con Google

[95]. Luni C, Giulitti S, Serena E, Ferrari L, Zambon A, Gagliano O, Giobbe GG, Michielin F, Knöbel S, Bosio A, Elvassore N. 2016. High-efficiency cellular reprogramming with microfluidics. Nature Methods 13:446-452. Cerca con Google

[96]. Elliott DA, Braam SR, Koutsis K, Nq ES, Jenny R, Lagerqvist EL, Biben C, Hatzistavrou T, Hirst CE, Yu QC, Skelton RJP, Ward-van Oostwaard D, Lim SM, Khammy O, Li X, Hawes SM, Davis RP, Goulburn AL, Passier R, Prall OWJ, Haynes JM, Pouton CW, Kaye DM, Mummery CL, Elefanty AG, Stanley EG. 2011. NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nature Methods 8:1037-1040. Cerca con Google

[97]. Zhang J, Klos M, Wilson GF, Herman AM, Lian X, Raval KK, Kamp TJ. 2012. Extracellular Matrix Promotes Highly Efficient Cardiac Differentiation of Human Pluripotent Stem Cells: The Matrix Sandwich Method. Circulation Research 111:1125-1136. Cerca con Google

[98]. Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, Wei S, Hao W, Kilgore J, William MS, Roth MG, Amatruda JF, Chen C, Lum F. 2009. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nature Chemical Biology 5:100-107. Cerca con Google

[99]. Saggin L, Ausoni S, Gorza L, Sartore S, Schiaffino S. 1988. Troponin T switching in the developing rat heart. Journal of Biological Chemistry 263:18488-18492. Cerca con Google

[100]. Saggin L, Gorza L, Ausoni S, Schiaffino S. 1989. Troponin I switching in the developing heart. Journal of Biological Chemistry 264:16299-16302. Cerca con Google

[101]. Lin X, Zemlin C, Hennan J, Petersen JS, Veenstra RD. 2008. Enhancement of Ventricular Gap Junction Coupling by Rotigaptide. Cardiovascular Research 79:416-426. Cerca con Google

[102]. van der Velden HM, Jongsma HJ. 2002. Cardiac gap junctions and connexins: their role in atrial fibrillation and potential as therapeutic targets. Cardiovascular Research 54:270-279. Cerca con Google

[103]. Bruzzone R, White TW, Paul DL. 1996. Connections with connexins: the molecular basis of direct intercellular signaling. European Journal of Biochemistry 238:1-27. Cerca con Google

[104]. Postma S, Rook MB, Jongsma HJ. 2001. Gap junctions in the rabbit sinoatrial node. American Journal of Physiology - Heart and Circulatory Physiology 280:2103-2115. Cerca con Google

[105]. Sakmann B, Neher E. 1984. Patch clamp techniques for studying ionic channels in excitable membranes. Annual Review of Physiology 46:455-472. Cerca con Google

[106]. Hamill OP, Marty A, Neher E, Sakmann B, Sihworth FJ. 1981. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pfluger Archiv European Journal of Physiology. 391:85-100. Cerca con Google

[107]. Karmazinova M, Lacinovà L. 2010. Measurement of Cellular Excitability by Whole Cell Patch Clamp Technique. Physiology Research 59:S1-S7. Cerca con Google

[108]. Hernandez V, Bortolozzi M, Pertegato V, Beltramello M, Giarin M, Zaccolo M, Pantano S, Mammano F. 2007. Unitary permeability of gap junction channels to second messengers measured by FRET microscopy and dual whole-cell current recordings. Nature Methods 4:353-358. Cerca con Google

[109]. White RL, Doeller JE, Verselis VK, Wittenberg BA. 1990. Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++. The Journal of General Physiology 95:1061-1075. Cerca con Google

[110]. Sackmann EK, Fulton AL, Beebe DJ. 2014. The present and future role of microfluidics in biomedical research. Nature 507:181-189. Cerca con Google

[111]. Beebe DJ, Glennys A, Walker MG, Walker M. 2002. Physics and Applications of Microfluidics in Biology. Annual Review of Biomedical Engineering 4:261-286. Cerca con Google

[112]. Tehranirokh M, Kouzani AZ, Francis PS, Kanwar JR. 2013. Microfluidic devices for cell cultivation and proliferation. Biomicrofluidics 7:051502-1-051502-32. Cerca con Google

[113]. Reyes DR, Iossifidis D, Aurox PA, Manz A. 2002. Micro total analysis systems. Introduction, theory, and technology. Analytical Chemistry 74:2623-2636. Cerca con Google

[114]. Le Gac S, van den Berg A. 2010. Single cells as experimentation units in lab-on-a-chip devices. Trends Biotechnol 28:55-62. Cerca con Google

[115]. Ranga A, Lutolf MP. 2012. High-throughput approaches for the analysis of extrinsic regulators of stem cell fate. Curr Opin Cell Biol 24:236-244. Cerca con Google

[116]. Rouwkema J, Rivron NC, van Blitterswijk CA. 2008. Vascularization in tissue engineering. Trends Biotechnol 26:434-441. Cerca con Google

[117]. van der Meer AD, van der Berg A. 2012. Organs-on-chips: breaking the in vitro impasse. Integrative Biology 4:461-470. Cerca con Google

[118]. Luni C, Serena E, Elvassore N. 2014. Human-on-chip for therapy development and fundamental science. Current opinion in biotechnology 25:45-50. Cerca con Google

[119]. Derby B. 2012. Printing and prototyping of tissues and scaffolds. Science 338:921-926. Cerca con Google

[120]. Figallo E, Cannizzaro C, Gerecht S, Burdick JA, Langer R, Elvassore N, Vunjak-Novakovic G. 2007. Micro-bioreactor array for controlling cellular microenvironments. Lab on a Chip 7:710-719. Cerca con Google

[121]. Wan CW, Chung S, Kamm RD. 2011. Differentiation of Embryonic Stem Cells into Cardiomyocytes in a Compliant Microfluidic System. Annals of Biomedical Engineering 39:1840-1847. Cerca con Google

[122]. Huh D, Torisawa Y, Hamilton GA, Kim HJ, Ingber DE. 2012. Microengineered physiological biomimicry: Organs-on-Chips. Lab on a Chip 12:2156-2164. Cerca con Google

[123]. Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HJ, Ingber DE. 2010. Reconstituting Organ-Level Lung Functions on a Chip. Science 328:1662-1668. Cerca con Google

[124]. Young EWK, Beebe JD. 2010. Fundamentals of Microfluidic Cell Culture in Controlled Microenvironments. Chemical Society Review 39:1036-1048. Cerca con Google

[125]. Discher DE, Mooney DJ, Zandstra PW. 2009. Growth factors, matrices, and forces combine and control stem cells. Science 324:1673-1677. Cerca con Google

[126]. Yu H, Alexanderbc CM, Beebeac DJ. 2007. Understanding microchannel culture: parameters involved in soluble factor signaling. Lab on a Chip 7:726-730. Cerca con Google

[127]. Thomas PC, Raghavan SR, Forry SP. 2011. Regulating Oxygen Levels in a Microfluidic Device. Analytical Chemistry 83:8821-8824. Cerca con Google

[128]. Shamloo A, Xu H, Heilshorn S. 2012. Mechanisms of Vascular Endothelial Growth Factor-Induced Pathfinding by Endothelial Sprouts in Biomaterials. Tissue Engineering Part A 18:320-330. Cerca con Google

[129]. Ashe HL, Briscoe J. 2006. The interpretation of morphogen gradients. Development 133:385-394. Cerca con Google

[130]. Masuda S, Washizu M, Nanba T. 1989. Novel method of cell fusion in field constriction area in fluid integration circuit. IEEE Transactions on Industry Applications 25:732-737. Cerca con Google

[131]. Mehta G, Lee J, Cha W, Tung Y, Linderman JJ, Takayama S. 2009. Hard Top Soft Bottom Microfluidic Devices for Cell Culture and Chemical Analysis. Analytical Chemistry 81:3714-3722. Cerca con Google

[132]. Halldorsson S, Lucumi E, Gómez-Sjöberg R, Fleming RMT. 2015. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosensors and Bioelectronics 63:218-231. Cerca con Google

[133]. Chou HP, Thorsen T, Scherer A, Quake SR. 2000. Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography. Science 288:113-116. Cerca con Google

[134]. Heo YS, Cabrera LM, Song JW, Futai N, Tung Y, Smith GD, Takayama S. 2007. Characterization and Resolution of Evaporation-Mediated Osmolality Shifts That Constrain Microfluidic Cell Culture in Poly(dimethylsiloxane) Devices. Analytical Chemistry 79:1126-1134. Cerca con Google

[135]. Kim L, Toh Y, Voldman J, Yu H. 2007. Tutorial Review: A practical guide to microfluidic perfusion culture of adherent mammalian cells. Lab on a Chip 7:681-694. Cerca con Google

[136]. Giulitti S, Magrofuoco E, Prevedello L, Elvassore N. 2013. Optimal periodic perfusion strategy for long-term micorlfuidic cell culture. Lab on a Chip 13:4430-4441. Cerca con Google

[137]. Beebe DJ, Moore JS, Bauer JM, Yu Q, Liu RH, Devadoss C, Jo B. 2000. Functional hydrogel structures for autonomous flow control inside microfluidic channels. Nature 404:588-590. Cerca con Google

[138]. Berthier E, Beebe DJ. 2007. Flow rate analysis of a surface tension driven passive micropump. Lab on a Chip 7:1475-1478. Cerca con Google

[139]. Whitesides GM, Ostuni E, Shuichi T, Xingyu J, Donald EI. 2001. Soft Lythography in Biology and Biochemistry. Annual Review of Biomedical Engineering 3:335-373. Cerca con Google

[140]. Elveflow Plug&Play microfluidics website, Vai! Cerca con Google

[141]. Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, Gardner RL, McKay RD. 2007. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448:196-199. Cerca con Google

[142]. Evans SM, Yelon D, Conlon FL, Kirby ML. 2010. Myocardial Lineage Development. Circulation Research 107:1428-1444. Cerca con Google

[143]. Rozario T, DeSimone DW. 2010. The extracellular matrix in development and morphogenesis: A dynamic view. Dev Biol 341:126-140. Cerca con Google

[144]. Kali K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjien K. 2009.Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458:771-775. Cerca con Google

[145]. Yu J, Hu. K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA. 2009. Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science 324:797-801. Cerca con Google

[146]. Kim D, Kim C, Moon J, Chung Y, Chang M, Han B, Ko S, Cha EYKY, Lanza R, Kim K. 2009. Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins. Cell stem cell 4:472-476. Cerca con Google

[147]. Fusaki N, Ban H, Nishiyama A, Saeaki K, Hasegawa M. 2009. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings on the Japan Academy, Series B 85:348-362. Cerca con Google

[148]. Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, Panetta NJ, Chen ZY, Robbins RC, Kay MA, Longaker MT, Wu JC. 2010. A nonviral minicircle vector for deriving human IPS cells. Nature Methods 7:197-199. Cerca con Google

[149]. Keisuke O, Masato N, Hong H, Tomoko I, Yamanaka S. 2008. Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors. Science 322:949-953. Cerca con Google

[150]. Samedan LTD, Pharmaceutical Publisher website,; magazine 12, issue 151, article 2902. Vai! Cerca con Google

[151]. Yisraeli JK, Melton DA. 1989. Synthesis of long, capped transcripts in Vitro by SP6 and T7 RNA polymerases. Meth Enzymol 180:42-50. Cerca con Google

[152]. Kreiter S, Selmi A, Simon P, Koslowski M, Huber C, Türeci Ö, Sahin U. 2006. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108:4009-4017. Cerca con Google

[153]. Van den Bosch GA, Van Gluck E, Ponsaerts P, Nijs G, Lenjou M, Apers L, Kint I, Heyndrickx L, Vanham G, Van Bockstaele DR, Berneman ZN, Van Tendeloo VFI. 2006. Simultaneous Activation of Viral Antigen-specific Memory CD4+ and CD8+ T-cells Using mRNA-electroporated CD40-activated Autologous B-cells. Journal of Immunotherapy 29:512-523. Cerca con Google

[154]. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. 2004. Innate Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA. Science 303:1529-1531. Cerca con Google

[155]. Ellegast J, Kim S, Brzózka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M, Endres S, Hartmann G. 2006. 5'-Triphosphate RNA Is the Ligand for RIG-I. Science 314:994-997. Cerca con Google

[156]. Pinchlmair A, Schulz O, Tan CP, Näslund TJ, Liljeström P, Welber F, Reis e Sousa C. 2006. RIG-I-Mediated Antiviral Responses to Single-Stranded RNA Bearing 5'-Phosphates. Science 314:997-1001. Cerca con Google

[157]. Nallagatla SR, Bevilacqua PC. 2008. Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner. RNA 14:1201-1213. Cerca con Google

[158]. Nallagatla SR, Toroney R, Bevilacqua PC. 2008. A brilliant disguise for self RNA: 5’-end and internal modifications of primary transcripts suppress elements of innate immunity. RNA Biology 5:140-144. Cerca con Google

[159]. Symons JA, Alcami A, Smith GL. 1995. Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell 81:551-560. Cerca con Google

[160]. Lui KO, Zangi L, Chien KR. 2014. Cardiovascular regenerative therapeutics via synthetic paracrine factor modified mRNA. Stem Cell Research 13:693-704. Cerca con Google

[161]. Zangi L, Lui KO, von Gise A, Ma Q, Ebina W, Ptaszek LM, Spater D, Xu H, Tabebordbar M, Gorbatov R, Sena B, Nahrendof M, Briscoe DM, Li RA, Wagers AJ, Rossi DJ, Pu WT, Chien KR. 2013. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nature Biotechnology 31:898-907. Cerca con Google

[162]. Davis RL, Weintraub H, Lassar AB. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987-1000. Cerca con Google

[163]. Preskey D, Allison TF, Jones M, Mamchaoui K, Unger C. 2016. Synthetically modified mRNA for efficient and fast human iPS cell generation and direct transdifferentiation to myoblasts. Biochem Biophys Res Commun 473:743-751. Cerca con Google

[164]. Xie H, Ye M, Feng R, Graf T. 2004. Stepwise reprogramming of B cells into macrophages. Cell 117:663-676. Cerca con Google

[165]. Laiosa CV, Stadtfeld M, Xie H, de Andres-Aguayo L, Graf T. 2006. Reprogramming of Committed T Cell Progenitors to Macrophages and Dendritic Cells by C/EBPα and PU.1 Transcription Factors. Immunity 25:731-744. Cerca con Google

[166]. Szabo E, Rampalli S, Risueno RM, Schnerc A, Mitchell R, Fiebyg-Comin A, Levadoux-Martin M, Bhatia M. 2010. Direct convertion of human fibroblasts to multilineage blood progenitors. Nature 468:521-526. Cerca con Google

[167]. Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. 2008. In vivo reprograming of adult pancreatic exocrine cells to beta-cells. Nature 455:109-113. Cerca con Google

[168]. Huang P, He Z, Ji S, Sun H, Xiang D, Liu C, Hu Y, Wang X, Hui L. 2011. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475:386-389. Cerca con Google

[169]. Caiazzo M, Dell'Anno MT, Dvoretskova E, Lazarevic D, Taverna S, Leo D, Sotnikova TD, Menegon A, Roncaglia P, Colciago G, Russo G, Carninci P, Pezzoli G, Gainetdinov RR, Gustincich s, Dityatev A, Broccoli V. 2011. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476:224-227. Cerca con Google

[170]. Chen JX, Krane M, Deutsch M, Wang L, Rav-Acha M, Gregoire S, Engels MC, Rajarajan K, Karra R, Abel ED, Wu JC, Milan D, Wu SM. 2012. Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5. Circulation Research 111:50-55. Cerca con Google

[171]. Inagawa K, Miyamoto K, Yamakawa H, Muraoka N, Sadahiro T, Umei T, Wada R, Katsumata Y, Kaneda R, Nakade K, Kurihara C, Obata Y, Miyake K, Fukuda K, Ieda M. 2012. Induction of Cardiomyocyte-Like Cells in Infarct Hearts by Gene Transfer of Gata4, Mef2c, and Tbx5. Circulation Research 111:1147-1156. Cerca con Google

[172]. Nam YJ, Nam YJ, Luo X, Qi X, Tan W, Huang GN, Acharya A, Smith CL, Tallquist MD, Neilson EG, Hill JA, Bessel-Duby R, Olson EN. 2012. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature 485:599-604. Cerca con Google

[173]. Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Lui L, Conway SJ, Fu JD, Srivastava D. 2012. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485:593-598. Cerca con Google

[174]. Bakker ML, Boink GJJ, Boukens BJ, Verkerk AO, van den Boogaard M, van den Haan AD, Hoogaars WMH, Buermans HP, de Bakker JMT, Seppen J, Tan HL, Moorman AFM, 't Hoen PAC, Christoffels VM. 2012. T-box transcription factor TBX3 reprogrammes mature cardiac myocytes into pacemaker-like cells . Cardiovascular Research 94:439-449. Cerca con Google

[175]. Chan SS, Shi X, Toyama A, Arpke RW, Dandapat A, Iacovino M, Kang J, Le G, Hagen HR, Garry DJ, Kyba M. 2013. Mesp1 Patterns Mesoderm into Cardiac, Hematopoietic, or Skeletal Myogenic Progenitors in a Context-Dependent Manner. Cell stem cell 12:587-601. Cerca con Google

[176]. Perrino C, Rockman HA. 2006. GATA4 and the Two Sides of Gene Expression Reprogramming. Circulation Research 98:715-716. Cerca con Google

[177]. Chen C, Schwartz, R,J,. 1996. Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription. Molecular and Cellular Biology 16:6372-6384. Cerca con Google

[178]. Bi W, Drake CJ, Schwarz JJ. 1999. The transcription factor MEF2C-null mouse exhibits complex vascular malformations and reduced cardiac expression of angiopoietin 1 and VEGF. Developmental Biology 211:255-267. Cerca con Google

[179]. Hatcher CJ, Kim M, Mah CS, Goldstein MM, Wong B, Mikawa T, Basson CT. 2001. TBX5 Transcription Factor Regulates Cell Proliferation during Cardiogenesis. Dev Biol 230:177-188. Cerca con Google

[180]. Qin H, Diaz A, Blouin L, Lebbink RJ, Patena W, Tanbun P, LeProust EM, McManus MT, Song JS, Ramalho-Santos M. 2014. Systematic Identification of Barriers to Human iPSC Generation. Cell 158:449-461. Cerca con Google

[181]. Torchio E. Master Degree in Industrial Biotechnology, University of Padova, A.Y. 2013/2014. Riprogrammazione di cellule somatiche umane mediante mmRNA in piattaforme microfluidiche. Cerca con Google

[182]. Itzhaki I, Rapoport S, Huber I, Mizrahi I, Zwi-Dantsis L, Arbel G, Schiller J, Gepstein L. 2011. Calcium Handling in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes. PLoS ONE 6:e18037. Cerca con Google

[183]. Fu JD, Yu HM, Wang R, Liang J, Yang HT. 2006. Developmental regulation of intracellular calcium transients during cardiomyocyte differentiation of mouse embryonic stem cells. Acta Pharmacologica Sinica 27:901-910. Cerca con Google

[184]. Lee YK, Ng KM, Lai WH, Chan YC, Lau YM, Lian Q, Siu CW. 2011. Calcium Homeostasis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Stem Cell Reviews 7:976-986. Cerca con Google

[185]. Martewicz S, Serena E, Zambon A, Mongillo M, Elvassore N. 2012. Reversible alterations of calcium dynamics in cardiomyocytes during acute hypoxia transient in a microfluidic platform. Integrative Biology 4:153-164. Cerca con Google

[186]. Bers DM. 2002. Cardiac excitation-contraction coupling. Nature 415:198-205. Cerca con Google

[187]. Doppler SA, Deutsch M-, Lange R, Krane M. 2013. Cardiac regeneration: current therapies—future concepts. Journal of Thoracic Disease 5:683-697. Cerca con Google

[188]. Vuoristo S, Toivonen S, Weltner J, Mikkola M, Ustinov J. 2013. A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation. PLoS ONE 8:76205. Cerca con Google

[189]. Blazeski A, Zhu R, Hunter DW, Weinberg SH, Zambidis ET, Tung L. 2012. Cardiomyocytes derived from human induced pluripotent stem cells as models for normal and diseased cardiac electrophysiology and contractility. Prog Biophys Mol Biol 110:166-177. Cerca con Google

[190]. van Weered JH, Koshiba-Takeuchi K, Kwon C, Takeuchi JK. 2011. Epigenetic factors and cardiac development. Cardiovascular Research 91:203-211. Cerca con Google

Solo per lo Staff dell Archivio: Modifica questo record