Acute myocardial infarction (AMI) and post ischemic congestive heart failure (CHF) are the major causes of mortality and morbidity in the western world. Despite medical advances in the treatment of STEMI, still numerous patients with large infarct size experience pathogenic left ventricular remodeling, eventually leading to CHF. Recently, the use of mesenchymal stem cells (MSC) has been proposed as a new therapy to restore cardiac tissue damage occurring after an ischemic event. In particular, a study by Professor Gnecchi, the supervisor of this Master thesis, showed that MSC are able to repair myocardial damage through paracrine mechanisms, consisting in the release of soluble factors such as cytokines, growth factors and microvescicles that act on the resident cardiac cells exerting multiple benefic effects. Professor Gnecchi and his research group have demonstrated that MSC isolated from the amniotic membrane of the human placenta(A-MSC) exert more potent paracrine effects compared with those of MSC isolated from bone marrow of adult human donors. In particular, A-MSC exert powerful cytoprotective and proangiogenic effects and their injection in a rat ischemia-reperfusion experimental model led to a limited infarcted area and recovery of cardiac function. For these reasons, the laboratory is developing a project with the aim of translating the observed benefic effects of MSC from bench to bedside. In particular, we are developing an in vitro protocol for the isolation, expansion and production of clinical-grade conditioned medium, compliant to the standards of good manufacturing products (GMP) conditions. GMP practices exclude the use of animal derived components in any stage of the cell culture. Within this project, I tested the effects of human platelet lysate (LP) as main supplement of the growth medium of the A-MSC, alternative to the fetal bovine serum (FBS). A-MSC from 10 different amniotic membranes have been isolated, cultured in presence of LP, their growth was monitored and the secretome produced. To investigate if the cells maintain the stemness, according to the International Society for Cellular Therapy (ISCT) criteria, the capacity to differentiate in osteoblasts, chondroblasts and adipocytes has been valuated, as well as the expression of specific membrane antigens through cytofluorimetry; and in addition the karyotype has been analyzed to exclude the presence of any chromosomic abnormalities. Subsequently, having established the identity of the A-MSC and their genomic integrity, we verified if the secretome had remained functional, as previously observed in the FBS cultured A-MSC. The cytoprotective effect of the conditioned medium was tested on rat cardiomyoblasts (H9c2) exposed to hypoxia and reoxygenation, to mimic an ischemic event in vitro, while the pro angiogenic capacity was studied using angiogenic assay with human umbilical vein endothelial cells. My results show that the A-MSC expanded in the presence of human LP satisfy the ISCT criteria to be defined as mesenchymal stem cells: they have the appropriate membrane antigens expression profile and are capable to differentiate in osteoblasts adipocytes and chondroblasts. Furthermore, no chromosomic abnormalities was observed, as shown by the karyotype analysis. The conditioned medium collected from the A-MSC cultured with human LP exerted pro angiogenic effect and cytoprotective actions similar with those previously obtained with the secretome of MSC cultured in FBS. In conclusion, the results collected show that the translation of A-MSC to an in vitro culture depleted of xenogen products do not modify the cells characteristics nor the cytoprotective or the proangiogenic properties of their conditioned medium.
L’infarto miocardico acuto (IMA) e l’insufficienza cardiaca congestizia (ICC) post ischemica sono tra le principali cause di mortalità e morbilità nel mondo occidentale. Nonostante i progressi nelle terapie mediche e chirurgiche, nella maggior parte dei pazienti con IMA di grandi dimensioni subentra un processo di rimodellamento ventricolare patologico che conduce a ICC. La terapia cellulare con cellule staminali mesenchimali (MSC) è stata proposta come nuova strategia per riparare il tessuto cardiaco danneggiato in seguito ad un evento ischemico. In particolare, ci sono chiare evidenze secondo cui il le MSC favoriscono la riparazione cardiaca mediante meccanismi paracrini, ovvero il rilascio di fattori solubili quali citochine, fattori di crescita e microvescicole che agiscono sulle cellule cardiache residenti esercitando diversi effetti benefici. Il Professor Gnecchi, assieme al suo gruppo di ricerca, ha dimostrato che MSC isolate dal foglietto amniotico della placenta (A-MSC) esercitano effetti paracrini più potenti rispetto a quelli delle MSC isolate da midollo osseo di adulti. In particolare, le A-MSC esercitano un potente effetto citoprotettivo e pro-angiogenico e la loro somministrazione in un modello sperimentale di ratto sottoposto a ischemia e riperfusione porta ad una riduzione della dimensione dell’infarto e miglioramento funzionale. Per questa ragione è stato intrapreso un progetto per traslare gli effetti benefici osservati con queste cellule dal laboratorio al letto del paziente. È in corso lo sviluppo di un protocollo per l’isolamento, l’espansione e la produzione di mezzo condizionato su larga scala secondo le norme di buona fabbricazione per la clinica (GMP, clinical-grade), che tra le varie cose, non prevedono componenti animali in nessuna fase della coltura cellulare. All’interno di questo progetto mi sono occupata di valutare gli effetti del lisato piastrinico umano (LP) come supplemento del terreno di coltura delle A-MSC, alternativo al siero bovino fetale (FBS). Per far ciò sono state isolate le A-MSC da 10 diverse placente, mantenute in coltura in presenza di LP, e ne è stata monitorata la crescita e la produzione del secretoma. Per ricercare se mantenessero le caratteristiche di cellule staminali, secondo i criteri della International Society for Cellular Therapy (ISCT) è stato eseguito il test di differenziamento in senso adipogenico, condrogenico e osteogenico, e si è verificata l’espressione di specifici antigeni di membrana tramite citofluorimetria; inoltre è stato analizzato il cariotipo per escludere la presenza di eventuali anomalie cromosomiche. Dopo aver accertato l’identità e l’integrità genomica delle A-MSC coltivate in LP, si è verificato che anche il secretoma fosse rimasto funzionale, come precedentemente osservato con A-MSC coltivate in FBS. La capacità citoprotettiva del loro mezzo condizionato è stata testata su cardiomioblasti di ratto sottoposti a saggio di ipossia e ri-ossigenazione, che mima in vitro un evento ischemico, mentre la capacità pro-angiogenica è stata valutata mediante saggio di angiogenesi con cellule endoteliali umane da vena ombelicale. I risultati dimostrano che le A-MSC espanse in presenza di LP sono conformi ai criteri dell’ISCT: hanno il corretto profilo di espressione degli antigeni di membrana, e sono state in grado di differenziare in osteoblasti, adipociti e condroblasti. Inoltre non sono state osservate anomalie cromosomiche come mostrato dal cariotipo effettuato. Il mezzo condizionato raccolto dalle A-MSC espanse in presenza di LP ha un effetto pro-angiogenico e ha un’azione citoprotettiva comparabile a quanto osservato in precedenza con cellule cresciute in FBS. In conclusione i miei dati dimostrano che la traslazione delle A-MSC ad una coltura priva di prodotti xenogeni non modifica le caratteristiche delle cellule né le proprietà citoprotettive o angiogeniche del loro mezzo condizionato.
Sviluppo di un protocollo clinical-grade per l’isolamento di cellule staminali mesenchimali di origine placentare e la produzione di mezzo condizionato per il trattamento della cardiopatia ischemica
NYKJAER, GIULIA
2018/2019
Abstract
Acute myocardial infarction (AMI) and post ischemic congestive heart failure (CHF) are the major causes of mortality and morbidity in the western world. Despite medical advances in the treatment of STEMI, still numerous patients with large infarct size experience pathogenic left ventricular remodeling, eventually leading to CHF. Recently, the use of mesenchymal stem cells (MSC) has been proposed as a new therapy to restore cardiac tissue damage occurring after an ischemic event. In particular, a study by Professor Gnecchi, the supervisor of this Master thesis, showed that MSC are able to repair myocardial damage through paracrine mechanisms, consisting in the release of soluble factors such as cytokines, growth factors and microvescicles that act on the resident cardiac cells exerting multiple benefic effects. Professor Gnecchi and his research group have demonstrated that MSC isolated from the amniotic membrane of the human placenta(A-MSC) exert more potent paracrine effects compared with those of MSC isolated from bone marrow of adult human donors. In particular, A-MSC exert powerful cytoprotective and proangiogenic effects and their injection in a rat ischemia-reperfusion experimental model led to a limited infarcted area and recovery of cardiac function. For these reasons, the laboratory is developing a project with the aim of translating the observed benefic effects of MSC from bench to bedside. In particular, we are developing an in vitro protocol for the isolation, expansion and production of clinical-grade conditioned medium, compliant to the standards of good manufacturing products (GMP) conditions. GMP practices exclude the use of animal derived components in any stage of the cell culture. Within this project, I tested the effects of human platelet lysate (LP) as main supplement of the growth medium of the A-MSC, alternative to the fetal bovine serum (FBS). A-MSC from 10 different amniotic membranes have been isolated, cultured in presence of LP, their growth was monitored and the secretome produced. To investigate if the cells maintain the stemness, according to the International Society for Cellular Therapy (ISCT) criteria, the capacity to differentiate in osteoblasts, chondroblasts and adipocytes has been valuated, as well as the expression of specific membrane antigens through cytofluorimetry; and in addition the karyotype has been analyzed to exclude the presence of any chromosomic abnormalities. Subsequently, having established the identity of the A-MSC and their genomic integrity, we verified if the secretome had remained functional, as previously observed in the FBS cultured A-MSC. The cytoprotective effect of the conditioned medium was tested on rat cardiomyoblasts (H9c2) exposed to hypoxia and reoxygenation, to mimic an ischemic event in vitro, while the pro angiogenic capacity was studied using angiogenic assay with human umbilical vein endothelial cells. My results show that the A-MSC expanded in the presence of human LP satisfy the ISCT criteria to be defined as mesenchymal stem cells: they have the appropriate membrane antigens expression profile and are capable to differentiate in osteoblasts adipocytes and chondroblasts. Furthermore, no chromosomic abnormalities was observed, as shown by the karyotype analysis. The conditioned medium collected from the A-MSC cultured with human LP exerted pro angiogenic effect and cytoprotective actions similar with those previously obtained with the secretome of MSC cultured in FBS. In conclusion, the results collected show that the translation of A-MSC to an in vitro culture depleted of xenogen products do not modify the cells characteristics nor the cytoprotective or the proangiogenic properties of their conditioned medium.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/21136