Hintergrund: Der medizinische Fortschritt der letzten Jahrzehnte verbessert das Überleben insbesondere extrem kleiner Frühgeborener. Bei diesen stellt die adäquate Oxygenierung über die unreife Lunge den kritischsten Prozess in der klinischen Betreuung dar, an dessen Ende häufig eine Beeinträchtigung der postnatalen Lungenentwicklung und -reifung steht. Klinisch als Bronchopulmonale Dysplasie (BPD) imponierend, stellt diese Erkrankung die häufigste Folge der extremen Frühgeburtlichkeit dar und ist im weiteren Verlauf mit einer bedeutenden Langzeitmortalität und gesundheitsökonomischen Belastung verbunden. Außer der Vermeidung der Frühgeburtlichkeit existiert keine Therapie für BPD. Exogene Mesenchymale Stromazellen (MSC) erwiesen sich jedoch in Tiermodellen der BPD als therapeutisch ausgesprochen wirksam und stellen somit einen vielversprechenden Therapieansatz dar. Dennoch ist wenig über die Mechanismen der exogenen MSC-Wirkung in der frühgeborenen Lunge bekannt; ein Verständnis dieser ist jedoch unabdingbar für eine sichere und effektive Translation von MSC-basierten Therapien in die klinische Anwendung. Hypothese: Lungenresidente MSC sind an der normalen Lungenentwicklung beteiligt, werden durch Bedingungen, welche die zu frühe Geburt simulieren, beeinträchtigt, und tragen so zur Pathogenese der BPD bei. Exogene MSC unterstützen die lungenresidenten MSC in ihrer normalen Funktion und/oder schützen sie vor Schaden. Methoden und Resultate: Um mesenchymale Zellen aus human-fetalem Lungengewebe (FLMSC) zu isolieren, wurde eine Methode zur enzymatischen Gewebedissoziation mit anschließender selektiver Dichtegradientenzentrifugation entwickelt. Der überwiegende Mehrheit der isolierten Lungenmesenchymzellen wurde als MSC identifiziert. Damit ist mit der hier vorliegenden Arbeit erstmals die vollständige Beschreibung von humanen, fetalen Lungen-MSC gelungen. Nabelschnur (UC)MSC wurden durch enzymatischen Verdau der Wharton-Sulze gewonnen. Kultur der FLMSC in einer hypoxischen, den intrauterinen Bedingungen ähnlichen Atmosphäre resultierte in einem das Lungenwachstum stimulierenden Cytokin- und Genexpressionsmuster. Zudem produzierten die FLMSC für Lungenwachstum und -reifung unabdingbare Extrazellulärmatrixproteine. Unter Exposition gegenüber hyperoxischen Kulturbedingungen – welche die zu frühe Geburt mit anschließender Behandlung auf einer Neugeborenenintensivstation simulierten – begannen FLMSC einen Transdifferenzierungsprozess und sezernierten proinflammatorische und antiangiogene Signalmoleküle. Zudem wurde eine Reduktion der Produktion von für die Lungenentwicklung unabdingbaren Matrixproteinen beobachtet. FLMSC sendeten zudem “Danger-Signale” an andere Zellen, sobald sie Hyperoxie ausgesetzt wurden. Exogene UCMSC sezernierten in vitro große Mengen an Proteinen, welche Lungenzellen vor Schaden schützen und Lungenwachstum und -differenzierung unterstützen. Diskussion und Schlussfolgerung: Das Mesenchym der humanen fetalen Lunge am Ende der kanalikulären Entwicklungsphase besteht zum Großteil aus MSC, und nicht Fibroblasten. Das impliziert eine mesenchymale Stamm- und Progenitorzellhierarchie in der fetalen Lunge sowie bislang unbeschriebene zelluläre mesenchymale Transdifferenzierungsprozesse im weiteren intrauterinen Entwicklungsverlauf. In vitro wurde Evidenz für eine Beteiligung der endogenen Lungen-MSC an der normalen Lungenentwicklung generiert; eine Beteiligung an der Koordination von epithelialem und endothelialem Lungenwachstum und -reifung durch die endogenen MSC kann auf Grund der vorliegenden Daten angenommen werden. Nach Exposition gegenüber Hyperoxie entwickelten FLMSC einen die BPD unterstützenden Phänotyp. Exogene UCMSC besitzen das Potential, die in diesem Zustand fehlenden Faktoren bereitzustellen. Endogene pulmonale MSC sind daher potentielles Ziel und potentielle Effektorzellpopulation einer MSC-basierten Therapie für BPD. Dennoch sind weitere in vivo Experimente mit Tiermodellen der extremen Frühgeburtlichkeit unabdingbar, um die Rolle der endogenen MSC in der normalen, und insbesondere gestörten Lungenentwicklung zu verstehen und folgend potente, und vor allem sichere zellbasierte Therapeutika für unsere wohl verletzlichste Patientenpopulation – Frühgeborene – bereitzustellen. / Background: Despite great achievements in neonatal and perinatal medicine over the past decades, the immature lung remains the most critical organ to care for after premature birth. As a consequence, impairment of of postnatal lung development – bronchopulmonary dysplasia or BPD – remains the most common complication of extreme prematurity and a major healthcare burden. There is no therapy for BPD, except prevention of premature birth. Recently, exogenous mesenchymal stromal cells (MSC) have been shown to prevent and rescue impaired lung development in animal models. Understanding the mechanisms behind the beneficial action of these cells is crucial for a successful, safe, and effective clinical translation of these promising MSC-based cell therapies in neonates. Hypothesis: Endogenous lung-resident MSC contribute to normal lung development and become impaired in conditions resembling premature birth, thus playing a part in the pathogenesis of BPD. Exogenous MSC act by supporting and/or preserving the endogenous mesenchymal cell’s function. Methods and Results: Using lung tissue from aborted fetuses, a novel enzyme/density gradient technique was employed to obtain endogenous human fetal lung mesenchymal cells (FLMSC). The vast majority of the so-isolated cells fulfilled all criteria of MSC, making the herein presented work the first complete description of MSC from human fetal lung tissue. Human umbilical cord-derived (UC)MSC were isolated by enzymatic digestion of the Wharton’s jelly. When cultured in hypoxic atmospheres resembling intrauterine conditions, resident FLMSC exerted a gene expression- and cytokine profile supporting epithelial and endothelial lung development, and secreted extracellular matrix components crucial for normal lung growth. After exposure to hyperoxia – thus mimicking premature birth and subsequent treatment on a neonatal intensive care unit – FLMSC showed signs of transdifferentiation, acquired a pro-inflammatory / anti-angiogeneitic secretory profile, diminished production of crucial extracellular matrix components and send out danger signals to other cells. Conversely, UCMSC secreted various paracrine factors protecting lung cells, and proteins contributing to lung growth and alveolarization. Discussion and Conclusions: The human fetal lung’s mesenchyme at the late canalicular stage of development mainly consists of MSC rather than fibroblasts, thus implying a complex mesenchymal stem-/progenitor cell hierarchy and previously undescribed cellular transdifferentiation processes of human endogenous lung mesenchymal progenitors during late pregnancy. Evidence for a contribution of FLMSC to normal lung development was generated in vitro, suggesting a co-ordination of endothelial and epithelial cell fate by human endogenous lung MSC. When challenged with hyperoxia, FLMSC cells acquire a phenotype contributing to the pathogenesis of the BPD. Conversely, UCMSC harbor the potential to provide the factors that these damaged resident MSC lack to produce. The endogenous MSC may therefore represent a potential target of cell-based therapies of BPD. However, in vivo data obtained from premature animals is inevitable to gain further insights into the contribution of endogenous lung MSC to normal and disrupted lung development and to clinically translate potent and safe MSC-based therapeutics for our most vulnerable patient population - premature infants.

Transforming growth factor beta 3 (TGF3) promotes tenogenic differentiation and may enhance tendon regeneration in vivo. This study aimed to apply TGF3 absorbed in decellularized equine superficial digital flexor tendon scaffolds, and to investigate the bioactivity of scaffold-associated TGF3 in an in vitro model. TGF3 could effectively be loaded onto tendon scaffolds so that at least 88% of the applied TGF3 were not detected in the rinsing fluid of the TGF3-loaded scaffolds. Equine adipose tissue-derived multipotent mesenchymal stromal cells (MSC) were then seeded on scaffolds loaded with 300 ng TGF3 to assess its bioactivity. Both scaffold-associated TGF3 and TGF3 dissolved in the cell culture medium, the latter serving as control group, promoted elongation of cell shapes and scaffold contraction (p < 0.05). Furthermore, scaffold-associated and dissolved TGF3 affected MSC musculoskeletal gene expression in a similar manner, with an upregulation of tenascin c and downregulation of other matrix molecules, most markedly decorin (p < 0.05). These results demonstrate that the bioactivity of scaold-associated TGF3 is preserved, thus TGF3 application via absorption in decellularized tendon scaffolds is a feasible approach.

A Doença Pulmonar Obstrutiva Crônica (DPOC) é caracterizada pela limitação persistente de trocas gasosas, usualmente progressiva e associada a uma resposta inflamatória crônica exacerbada das vias aéreas a partículas e gases nocivos. Apesar de prevenível e tratável, não se logrou até o presente uma terapêutica eficaz, que resulte na cura da doença. Neste cenário, a terapia celular apresenta-se como uma alternativa terapêutica potencialmente promissora em DPOC, bem como em outras doenças pulmonares degenerativas e de caráter inflamatório. Porém, vários aspectos da terapia celular carecem de um melhor entendimento. Um dos principais desafios ao sucesso da terapia celular são as baixas taxas de sobrevivência das células transplantadas. O Inibidor do Ativador de Plasminogênio 1 (Plasminogen Activator Inhibitor 1 - PAI-1) pode representar um potencial mediador da sobrevivência de células estromais mesenquimais (CTM) pós-transplante, pois tem sido proposto que anticorpos neutralizadores do PAI-1 auxiliam no aumento da sobrevivência de CTM no tecido-alvo da terapia celular. Desta forma, a diminuição dos níveis de PAI-1 possui um potencial terapêutico interessante, ao modular os principais processos envolvidos na criação de um ambiente pouco propício ao \"homing\" celular durante o processo de injúria. A diminuição dos níveis de PAI-1 é promovida, pela sinvastatina, fármaco da família das estatinas. Desta forma, objetivou-se com este trabalho analisar os efeitos da sinvastatina sobre a expressão do PAI-1, bem como sua influência na sobrevivência das células infundidas para terapia celular de enfisema pulmonar em modelo murino. Camundongos da linhagem FVB foram submetidos à instilação intranasal de elastase para indução de enfisema pulmonar e, posteriormente, tratados com CTM do tecido adiposo e sinvastatina. Os resultados mostraram que, quanto aos aspectos morfológicos e funcionais, considerando-se a análise conjunta de ambos os pulmões, não houve diferença estatisticamente significativa entre os grupos submetidos à instilação intranasal de elastase e submetidos à terapia celular com CTM tratados ou não com sinvastatina. Quando porém os pulmões foram analisados individualmente constatou-se que não houve diferença estatisticamente significativa entre os grupos controle e os resultados referentes ao lado direito do pulmão dos animais tratados com elastase e que receberam sinvastatina e infusão de CTM. Diferenças anatômicas entre os lados direito e esquerdo do pulmão, levaram a uma maior deposição de células no lado direito, como evidenciado pelos resultados obtidos nos ensaios de bioluminescência. Pode-se, portanto, inferir que a recuperação morfológica no lado direito do pulmão de animais com DPOC/enfisema poderia ser decorrente de um efeito regenerativo parácrino das CTM associadas à sinvastatina. / Chronic Obstructive Pulmonary Disease - COPD is characterized by the persistent limitation of gas exchange, is usually progressive, and associated to a chronic augmented inflammatory response of the airways to particles and noxious gases. Despite preventable e treatable, an effective, curative therapeutic approach is yet to be achieved. In this context, cell therapy presents itself as a promising therapeutic approach for COPD and other pulmonary inflammatory and degenerative diseases. However, many aspects of cell therapy with stem cells remain unclear. One of the major challenges to the success of cell therapy are the low survival rates of transplanted cells. The Plasminogen Activator Inhibitor 1 (PAI-1) is a potential mediator of the survival of mesenchymal stromal cells (MSC) after transplantation, since PAI-1 neutralizing antibodies have been shown to increase the survival rate of MSC in the target tissue of cell therapy. Thus, the decreased levels of PAI-1 has an interesting therapeutic potential to modulate key processes involved in creating an inhospitable environment, during the process of injury, to the homing of transplanted cells. Decreased levels of PAI-1 are promoted by simvastatin, a drug of the statins family. Thus, the goal of this work was to evaluate the effect of simvastatin in vivo on the expression of PAI-1, as well as its influence on the survival rate of infused cells in mice model of cell therapy for pulmonary COPD/emphysema. FVB mice were submitted pulmonary emphysema induction by means of intranasal instillation of elastase and than treated with adipose-derived mesenchymal stem cells and simvastatin. The results regarding morphological and functional aspects, when considering the analisys of both lungs, presented no statistically significant difference among the groups submitted to intranasal instillation of elastase and cell therapy with MSC, treated or not with simvastatin. However, when the lungs where analyzed individually, it was found that there was no statistically significant difference between the control group and the results regarding the right lung of animals treated with elastase and that received simvastatin and MSC infusion. Anatomical differences between the right and left sides of the lung lead to a higher deposition of cells in the right side, as observed in the bioluminescence assays. Thus, it is possible to infer that the morphological recovery in the right side of the lung of animals with DPOC/emphysema could be due to a regenerative paracrine effect of mesenchymal stem cells associated with simvastatin.

A Doença Pulmonar Obstrutiva Crônica (DPOC) é caracterizada pela limitação persistente de trocas gasosas, usualmente progressiva e associada a uma resposta inflamatória crônica exacerbada das vias aéreas a partículas e gases nocivos. Apesar de prevenível e tratável, não se logrou até o presente uma terapêutica eficaz, que resulte na cura da doença. Neste cenário, a terapia celular apresenta-se como uma alternativa terapêutica potencialmente promissora em DPOC, bem como em outras doenças pulmonares degenerativas e de caráter inflamatório. Porém, vários aspectos da terapia celular carecem de um melhor entendimento. Um dos principais desafios ao sucesso da terapia celular são as baixas taxas de sobrevivência das células transplantadas. O Inibidor do Ativador de Plasminogênio 1 (Plasminogen Activator Inhibitor 1 - PAI-1) pode representar um potencial mediador da sobrevivência de células estromais mesenquimais (CTM) pós-transplante, pois tem sido proposto que anticorpos neutralizadores do PAI-1 auxiliam no aumento da sobrevivência de CTM no tecido-alvo da terapia celular. Desta forma, a diminuição dos níveis de PAI-1 possui um potencial terapêutico interessante, ao modular os principais processos envolvidos na criação de um ambiente pouco propício ao \"homing\" celular durante o processo de injúria. A diminuição dos níveis de PAI-1 é promovida, pela sinvastatina, fármaco da família das estatinas. Desta forma, objetivou-se com este trabalho analisar os efeitos da sinvastatina sobre a expressão do PAI-1, bem como sua influência na sobrevivência das células infundidas para terapia celular de enfisema pulmonar em modelo murino. Camundongos da linhagem FVB foram submetidos à instilação intranasal de elastase para indução de enfisema pulmonar e, posteriormente, tratados com CTM do tecido adiposo e sinvastatina. Os resultados mostraram que, quanto aos aspectos morfológicos e funcionais, considerando-se a análise conjunta de ambos os pulmões, não houve diferença estatisticamente significativa entre os grupos submetidos à instilação intranasal de elastase e submetidos à terapia celular com CTM tratados ou não com sinvastatina. Quando porém os pulmões foram analisados individualmente constatou-se que não houve diferença estatisticamente significativa entre os grupos controle e os resultados referentes ao lado direito do pulmão dos animais tratados com elastase e que receberam sinvastatina e infusão de CTM. Diferenças anatômicas entre os lados direito e esquerdo do pulmão, levaram a uma maior deposição de células no lado direito, como evidenciado pelos resultados obtidos nos ensaios de bioluminescência. Pode-se, portanto, inferir que a recuperação morfológica no lado direito do pulmão de animais com DPOC/enfisema poderia ser decorrente de um efeito regenerativo parácrino das CTM associadas à sinvastatina. / Chronic Obstructive Pulmonary Disease - COPD is characterized by the persistent limitation of gas exchange, is usually progressive, and associated to a chronic augmented inflammatory response of the airways to particles and noxious gases. Despite preventable e treatable, an effective, curative therapeutic approach is yet to be achieved. In this context, cell therapy presents itself as a promising therapeutic approach for COPD and other pulmonary inflammatory and degenerative diseases. However, many aspects of cell therapy with stem cells remain unclear. One of the major challenges to the success of cell therapy are the low survival rates of transplanted cells. The Plasminogen Activator Inhibitor 1 (PAI-1) is a potential mediator of the survival of mesenchymal stromal cells (MSC) after transplantation, since PAI-1 neutralizing antibodies have been shown to increase the survival rate of MSC in the target tissue of cell therapy. Thus, the decreased levels of PAI-1 has an interesting therapeutic potential to modulate key processes involved in creating an inhospitable environment, during the process of injury, to the homing of transplanted cells. Decreased levels of PAI-1 are promoted by simvastatin, a drug of the statins family. Thus, the goal of this work was to evaluate the effect of simvastatin in vivo on the expression of PAI-1, as well as its influence on the survival rate of infused cells in mice model of cell therapy for pulmonary COPD/emphysema. FVB mice were submitted pulmonary emphysema induction by means of intranasal instillation of elastase and than treated with adipose-derived mesenchymal stem cells and simvastatin. The results regarding morphological and functional aspects, when considering the analisys of both lungs, presented no statistically significant difference among the groups submitted to intranasal instillation of elastase and cell therapy with MSC, treated or not with simvastatin. However, when the lungs where analyzed individually, it was found that there was no statistically significant difference between the control group and the results regarding the right lung of animals treated with elastase and that received simvastatin and MSC infusion. Anatomical differences between the right and left sides of the lung lead to a higher deposition of cells in the right side, as observed in the bioluminescence assays. Thus, it is possible to infer that the morphological recovery in the right side of the lung of animals with DPOC/emphysema could be due to a regenerative paracrine effect of mesenchymal stem cells associated with simvastatin.

[ES] Las células mesenquimales estromales (MSC) poseen una serie de cualidades inmunológicas, pro-angiogénicas y regenerativas que las convierten en un excelente candidato para el tratamiento de diversas patologías. A pesar de las pruebas contundentes obtenidas en modelos preclínicos que demuestran la actividad terapéutica de las MSC, los ensayos clínicos no han podido mostrar hasta ahora un beneficio consistente, probablemente debido a deficiencias metodológicas y a la falta de estandarización, así como a la variabilidad genética intrínseca a los estudios en humanos. Debido a ello, ha sido necesario profundizar en los mecanismos responsables del beneficio terapéutico y rediseñar las estrategias clínicas. En los últimos años, se ha observado que la reparación tisular mediada por las MSC se produce de forma paracrina, y se ha constatado que las vesículas extracelulares (EVs) secretadas por las MSC (EVMSC) son capaces de recapitular las propiedades inmunosupresoras de las células parentales. Además, las estrategias terapéuticas basadas en vesículas tienen grandes ventajas en términos de bioseguridad y producción en condiciones de grado clínico, reduciendo significativamente el coste de dichas terapias. Sin embargo, la dosis efectiva en grandes mamíferos, incluidos los humanos, es bastante elevada y la producción industrial de EVs se ve dificultada en parte, por la senescencia proliferativa que afecta a las MSC durante la expansión celular masiva. En este trabajo hemos intentado solventar los principales escollos de la terapia con EVs incrementando su potencial inmunosupresor y reduciendo por tanto la dosis efectiva. Este incremento se ha conseguido gracias a la sobreexpresión del factor inducible por hipoxia 1-alpha y al desarrollo de un medio de acondicionamiento en cultivo basado en citoquinas. Además, la inmortalización de las células secretoras mediante la transducción del gen de la telomerasa humana ha permitido tanto la estandarización del producto como su producción a gran escala. La eficacia de estas EVs ha sido testada en diferentes poblaciones celulares in vitro: linfocitos T, monocitos, células Natural Killer, macrófagos, células endoteliales y fibroblastos; y en dos modelos de ratón: hipersensibilidad retardada y colitis aguda inducida por TNBS. En conclusión, hemos desarrollado una fuente de EVs de larga duración que secreta grandes cantidades de vesículas con mayor capacidad inmunosupresora y antiinflamatoria, facilitando un producto terapéutico más estándar y fácil de producir para el tratamiento de enfermedades inflamatorias inmunomediadas. / [CA] Les cèl·lules mesenquimals estromals (MSC) posseeixen una sèrie de qualitats immunològiques, pro-angiogèniques i regeneratives que les converteixen en un excel·lent candidat per al tractament de diverses patologies. Tot i les proves contundents obtingudes en models preclínics que demostren l'activitat terapèutica de les MSC, els assaigs clínics no han pogut mostrar fins ara un benefici consistent, probablement degut a deficiències metodològiques i a la manca d'estandardització, així com a la variabilitat genètica intrínseca als estudis en humans. A causa d'això, ha calgut aprofundir en els mecanismes responsables del benefici terapèutic i redissenyar les estratègies clíniques. En els últims anys, s'ha observat que la reparació tissular intervinguda per les MSC es produeix de forma paracrina, i s'ha constatat que les vesícules extracel·lulars (EVs) secretades per les MSC (EVMSC) són capaços de recapitular les propietats immunosupressores de les cèl·lules parentals. A més, les estratègies terapèutiques basades en vesícules tenen grans avantatges en termes de bioseguretat i producció en condicions de grau clínic, reduint significativament el cost d'aquestes teràpies. No obstant això, la dosi efectiva en grans mamífers, inclosos els humans, és bastant elevada i la producció industrial de les EVs es veu dificultada en part, per la senescència proliferativa que afecta les MSC durant l'expansió cel·lular massiva. En aquest treball hem intentat solucionar els principals esculls de la teràpia amb EVs incrementant el seu potencial immunosupressor i reduint per tant la dosi efectiva. Aquest increment s'ha aconseguit gràcies a la sobreexpressió del factor induïble per hipòxia 1-alpha i a el desenvolupament d'un mitjà de condicionament en cultiu basat en citoquines. A més, la immortalització de les cèl·lules secretores mitjançant la transducció del gen de la telomerasa humana ha permès tant l'estandardització del producte com la seva producció a gran escala. L'eficàcia d'aquestes EVs ha estat testada en diferents poblacions cel·lulars in vitro: limfòcits T, monòcits, cèl·lules Natural Killer, macròfags, cèl·lules endotelials i fibroblasts; i en dos models de ratolí: hipersensibilitat retardada i colitis aguda induïda per TNBS. En conclusió, hem desenvolupat una font de EVs de llarga durada que secreta grans quantitats de vesícules amb major capacitat immunosupressora i antiinflamatòria, facilitant un producte terapèutic més estàndard i fàcil de produir per al tractament de malalties inflamatòries inmunomediades. / [EN] Mesenchymal stromal cells (MSC) possess several immunological, pro-angiogenic and regenerative qualities that make them an excellent candidate for the treatment of various pathologies. Despite compelling evidence from preclinical models demonstrating the therapeutic activity of MSCs, clinical trials have so far failed to show consistent benefit, probably due to methodological shortcomings and lack of standardisation, as well as the genetic variability intrinsic to human studies. As a result, it has been necessary to further investigate the mechanisms responsible for therapeutic benefit and to redesign clinical strategies. In recent years, it has been observed that MSC-mediated tissue repair occurs in a paracrine pathway, and it has been confirmed that extracellular vesicles (EVs) secreted by MSC (EVMSC) are able to recapitulate the immunosuppressive properties of the parental cells. Moreover, vesicle-based therapeutic strategies have great advantages in terms of biosafety and production under clinical-grade conditions, significantly reducing the cost of such therapies. However, the effective dose in large mammals, including humans, is quite high and the industrial production of EVs is hampered in part by the proliferative senescence that affects MSC during massive cell expansion. In this work, we have attempted to overcome the main challenges of EVs therapy by increasing their immunosuppressive potential and thus reducing the effective dose. This increase has been achieved by overexpression of hypoxia-inducible factor 1-alpha and the development of a cytokine-based culture conditioning medium. In addition, immortalization of secretory cells by transduction with the human telomerase gene has allowed both product standardisation and large-scale production. The efficacy of these EVs has been tested in different cell populations in vitro: T lymphocytes, monocytes, Natural Killer cells, macrophages, endothelial cells and fibroblasts; and in two mouse models: delayed-type hypersensitivity and TNBS-induced acute colitis. In conclusion, we have developed a long-lasting source of EVs that secretes large amounts of vesicles with enhanced immunosuppressive and anti-inflammatory capacity, providing a more standard and easier-to-produce therapeutic product for the treatment of immune-mediated inflammatory diseases. / Gómez Ferrer, M. (2022). Terapias innovadoras basadas en vesículas extracelulares derivadas de células madre mesenquimales modificadas genéticamente [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182560 / TESIS

[ES] Actualmente la insuficiencia cardiaca es una de las principales causas de mortalidad y co-morbilidad a nivel mundial sobre todo en países desarrollados. Este tipo de patología a su vez, se desglosa en un gran abanico de posibles agentes que perturban el funcionamiento del músculo cardiaco en función de su causa. Uno de los problemas más importantes a niveles de afectación cardiaca se centra en pacientes que sufren algún tipo de cáncer y son tratados con antraciclinas. Estas drogas ampliamente usadas en el mundo de la oncología, tienen una serie de efectos secundarios entre los cuales destaca su posible efecto cardiotóxico. Este trabajo ahonda sobre este proceso y sobre la existencia de un perfil de paciente con una susceptibilidad a sufrir este tipo de episodios de toxicidad cardiaca. En este sentido, se investiga una serie de miRNAs que se encuentran diferencialmente expresados en pacientes que sufren este tipo de episodios frente a los que no. Una vez se detectan estos miRNA generamos un algoritmo predictor mediante el cual somos capaces de predecir si un paciente sufrirá cardiotoxicidad en función de la cantidad de ciertos miRNAs presentes en suero. Una vez descubiertos estos miRNAs nos centramos en la búsqueda de una posible opción terapéutica para esta serie de eventos. Para ello usamos las vesículas extracelulares (EVs) derivadas de células mesenquimales estromales (MSC), unas células que han demostrado tener un alto potencial terapéutico y de gran seguridad. Se evaluó su capacidad terapéutica en el daño inducido por doxorrubicina y posteriormente se amplió el estudio añadiendo el daño por isquemia reperfusión en diferentes frentes como puede ser la regeneración cardiaca, inhibición de la fibrosis o neoangiogénesis. Aunque es necesario un estudio más exhaustivo de los perfiles de cardiotoxicidad y de los mecanismos de acción tanto de los miRNAs como de las EVs, esta tesis demuestra la capacidad de ambos como una potente herramienta tanto de diagnóstico como terapéutica. / [CA] Actualment la insuficiència cardíaca és un dels principals causes de mortalitat i co-morbiditat a nivell mundial sobretot en països desenvolupats. Aquest tipus de patologia al seu torn, es desglossa en un gran ventall de possibles agents que pertorben el funcionament de l'múscul cardíac en funció de la seva causa. Un dels problemes més importants a nivells d'afectació cardíaca se centra en pacients que pateixen algun tipus de càncer i són tractats amb antraciclines. Aquestes drogues àmpliament usades en el món de l'oncologia, tenen una sèrie d'efectes secundaris entre els quals destaca el seu possible efecte cardiotòxic. Aquest treball aprofundeix sobre aquest succés i sobre ha un perfil de pacient amb una susceptibilitat a patir aquest tipus d'episodis de toxicitat cardíaca. En aquest sentit s'investiga una sèrie de miRNAs que es troben diferencialment expressats en pacients que pateixen aquest tipus d'episodis enfront dels que no. Un cop es detecten aquests miRNA generem un algoritme predictiu mitjançant el qual, som capaços de predir si un pacient patirà cardiotoxicitat en funció de la quantitat de certs miRNAs presents en sèrum. Un cop descoberts aquests miRNAs ens centrem en la recerca d'una possible opció terapèutica per a aquesta sèrie d'esdeveniments. Per a això fem servir les vesícules extracelulres derivades de cèl·lules mesesnquimales, unes cèl·lules que han demostrat tenir un alt potencial terapèutico i de gran seguretat. Es testen la seva capacitat terapèutica en el dany per doxurbicina i posteriorment s'amplia l'estudi afegint el dany per isquèmia reperfusió en diferents fronts com pot ser la regeneració cardíaca, inhibició de la fibrosi o neoangiogènesi. Encara que és necessari un estudi més exhaustiu dels perfils de cardiotoxicitat i dels mecanismes d'acció tant dels miRNAs com de les EVs, aquesta tesi demostra la capacitat de tots dos com una potent eina tant de diagnòstic com terapèutica. / [EN] Heart failure is currently one of the main causes of mortality and co-morbidity worldwide, especially in developed countries. This type of pathology, can be broken down into a wide range of possible agents that disturb the functioning of the cardiac muscle depending on its cause. One of the most important problems in terms of cardiac involvement is centered on patients who suffer from some type of cancer and are treated with anthracyclines. These drugs, widely used in the world of oncology, have a series of side effects, among which their possible cardiotoxic effect stands out. This work delves into this event and into the existence of a patient profile with a susceptibility to suffer this type of episodes of cardiac toxicity. In this sense, we investigate a series of miRNAs that are differentially expressed in patients who suffer this type of episodes versus those who do not. Once these miRNAs were detected, we generated a predictive algorithm by which we are able to predict whether a patient will suffer cardiotoxicity based on the amount of certain miRNAs present in serum. Once these miRNAs were discovered, we focused on the search for a possible therapeutic option for this series of events. For this we used extracellular vesicles (EVs) derived from mesenchymal cells (MSC), cells that have been shown to have a high therapeutic potential and high safety. We tested their therapeutic capacity in doxorubicin injury and later extended the study by adding ischemia reperfusion injury on different fronts such as cardiac regeneration, inhibition of fibrosis or neoangiogenesis. Although a more exhaustive study of the cardiotoxicity profiles and mechanisms of action of both miRNAs and EVs is needed, this thesis demonstrates the capacity of both as a powerful diagnostic and therapeutic tool. / Sánchez Sánchez, R. (2021). Estudio del papel de los miRNAs y las vesículas extracelulares derivadas de células mesenquimales estromales en la patología cardiaca [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/177178 / TESIS

Transplantation of multipotent mesenchymal progenitor cells is a valuable option for treating tendon disease. Tenogenic differentiation leading to cell replacement and subsequent matrix modulation may contribute to the regenerative effects of these cells, but it is unclear whether this occurs in the inflammatory environment of acute tendon disease. Equine adipose-derived stromal cells (ASC) were cultured as monolayers or on decellularized tendon scaffolds in static or dynamic conditions, the latter represented by cyclic stretching. The impact of different inflammatory conditions, as represented by supplementation with interleukin-1β and/or tumor necrosis factor-α or by co-culture with allogeneic peripheral blood leukocytes, on ASC functional properties was investigated. High cytokine concentrations increased ASC proliferation and osteogenic differentiation, but decreased chondrogenic differentiation and ASC viability in scaffold culture, as well as tendon scaffold repopulation, and strongly influenced musculoskeletal gene expression. Effects regarding the latter differed between the monolayer and scaffold cultures. Leukocytes rather decreased ASC proliferation, but had similar effects on viability and musculoskeletal gene expression. This included decreased expression of the tenogenic transcription factor scleraxis by an inflammatory environment throughout culture conditions. The data demonstrate that ASC tenogenic properties are compromised in an inflammatory environment, with relevance to their possible mechanisms of action in acute tendon disease.

Matrix remodeling could be an important mode of action of multipotent mesenchymal stromal cells (MSC) in extracellular matrix (ECM) disease, but knowledge is limited in this respect. As MSC are well-known to adapt their behavior to their environment, we aimed to investigate if their mode of action would change in response to healthy versus pathologically altered ECM. Human MSC-derived ECM was produced under different culture conditions, including standard culture, culture on Matrigel-coated dishes, and stimulation with the pro-fibrotic transforming growth factor-1 (TGF1). The MSC-ECM was decellularized, characterized by histochemistry, and used as MSC culture substrate reflecting different ECM conditions. MSC were cultured on the different ECM substrates or in control conditions for 2 days. Culture on ECM increased the presence of surface molecules with ECM receptor function in the MSC, demonstrating an interaction between MSC and ECM. In MSC cultured on Matrigel-ECM and TGF1-ECM, which displayed a fibrosis-like morphology, gene expression of collagens and decorin, as well as total matrix metalloproteinase (MMP) activity in the supernatant were decreased as compared with control conditions. These results demonstrated that MSC adapt to their ECM environment, which may include pathological adaptations that could compromise therapeutic efficacy.

Mesenchymale Stromazellen (MSC) unterstützen die endogene Geweberegeneration und sind kaum immunogen. Die Mechanismen der Immunmodulation sind kaum bekannt. Diese Arbeit untersucht, ob MSC in die Interaktion von Dendritischen Zellen (DC), Natürlichen Killer (NK) und T Zellen eingreifen, indem sie die DC-Reifung beeinflussen. Das Netzwerk ist wichtig für die Differenzierung naïver T Zellen zu Typ 1 T Helferzellen (Th1). Abhängig vom DC-Subtyp und dem Zeitpunkt des Aufeinandertreffens, beeinflussten Knochenmark-MSC (BM-MSC) die in vitro DC-Reifung verschieden. Sie inhibierten die Differenzierung, aber nicht die Reifung humaner von Monozyten-abgeleiteter DC (moDC). BM MSC hatten keinen klaren Einfluss auf die Reifung plasmazytoider DC (pDC), während sie in aktivierten CD1c+ myeloiden DC (mDC) einen tolerogenen Phänotyp induzierten, charakterisiert durch eine geringere CCR7-abhängige Migration und ein tolerogenes Zytokinprofil. Daraus resultierend, wiesen BM-MSC-geprägte mDC aufgrund der veränderten IL 12/IL 10 Sekretion eine geringere Fähigkeit zur Stimulation der IFNγ Produktion in NK Zellen auf und induzierten weniger Th1 Differenzierung naïver T Zellen. Placenta-derived mesenchymal-like adherent stromal cells (PLX PAD) erzielten ähnliche Ergebnisse. Es konnte keine Alloimmunogenität in Patienten mit kritischer Ischämie der Extremitäten (CLI), die im Rahmen einer Phase I klinischen Studie allogene PLX PAD erhalten hatten, nachgewiesen werden. Keiner der Patienten entwickelte eine signifikante Gedächtnis T Zellantwort spezifisch für das Zellprodukt, was durch unsere in vitro Beobachtungen erklärbar sein könnte. Es ist schwierig MSC im Gewebe nachzuweisen, da Markerkombinationen notwendig sind. CD73+CD90+CD105+CD45-CD34-CD14-CD19- MSC konnten mithilfe einer neuen Multiplex-Immunhistologie-Technik (Chipzytometrie) in humanen Plazentaschnitten detektiert werden. Für die Zukunft könnte damit die Interaktion injizierter MSC mit Immunzellen in Biopsien untersucht werden. / Mesenchymal stromal cells (MSC) support endogenous tissue regeneration and seem to be low immunogenic, allowing application across MHC barriers. But little is known about the mechanisms for their immunomodulation. Hence, the main goal of this study was to understand if MSC interfere with the crosstalk between dendritic cells (DC), natural killer (NK) and T cells by influencing DC maturation. This network is important for efficient priming of naïve T cells into type 1 helper T cells (Th1). Bone marrow-derived MSC (BM-MSC) had diverse effects on DC maturation in vitro, depending on the DC subset and the time of interaction. BM MSC inhibited differentiation but not maturation of monocyte-derived DC (moDC). They did not have a clear effect on maturation of plasmacytoid DC (pDC), whereas they induced a tolerogenic phenotype in activated CD1c+ myeloid DC (mDC), characterized by an impaired CCR7-dependent migration and a tolerogenic cytokine profile. Consequently, BM-MSC-licensed mDC displayed a reduced ability to induce IFNγ production in NK cells due to their altered IL 12/IL 10 secretion. BM MSC-licensed mDC also induced less efficiently Th1 lineage commitment of naïve T cells. Similar results were observed with placenta-derived mesenchymal-like adherent stromal cells (PLX PAD). Samples from critical limb ischemia (CLI) patients treated with MHC-unmatched PLX-PAD within a phase I clinical trial were analysed for alloimmunogenicity. None of the patients developed a significant memory T cell response specific to the allogeneic cells, which might be explainable by our in vitro observations. MSC are difficult to detect in tissues because a set of lineage markers is needed. Here, CD73+CD90+CD105+CD45-CD34-CD14-CD19- MSC could be identified in human placenta cryosections using a novel multiplex-immunohistology technique (chipcytometry), offering the possibility to investigate the crosstalk between injected MSC and attracted immune cells in patient biopsies in the future.