Dissertations / Theses on the topic 'Menstrual fluid stem cells'

INTRODUÇÃO: As células-tronco mesenquimais são capazes de regenerar diferentes tipos de tecidos, no entanto, a maioria dos métodos para sua obtenção são invasivos. Recentemente, foi descoberta a existência destas células no sangue menstrual. OBJETIVO: Padronizar as técnicas de coleta e transporte do fluido menstrual, bem como a caracterização, isolamento, expansão e criopreservação de células-tronco do fluido menstrual e avaliar a disponibilidade de células tronco mesenquimais no fluido menstrual. MÉTODOS: No período de agosto de 2011 a março de 2012 foram selecionadas 20 voluntárias com ciclo menstrual regular, sem doença ginecológica. O fluido menstrual foi coletado no dia de maior fluxo e submetido a imunofenotipagem e cultivo celular. Foram realizadas duas passagens em meio de cultura até atingir semi-confluência das células-tronco, as quais foram, em seguida, criopreservadas. RESULTADOS: Os parâmetros analisados apresentaram os seguintes valores médios: volume de fluido menstrual 6,90±5,60mL; tempo de transporte 17,20±5,50h; número de células totais 3,95 x106±3,88 x106 com 76,05%±24,57 de células viáveis. Após a cultura, as células mesenquimais aumentaram de 0,14%±0,26 para 96,19%±2,14. Na primeira passagem de cultura, após 15 a 21 dias, as colônias formaram grupos que atingiram a confluência, que a partir da segunda passagem ocorreu em cerca de 3 dias. As células-tronco mesenquimais criopreservadas eram viáveis. CONCLUSÃO: As células-tronco do fluido menstrual podem ser obtidas sem métodos invasivos. O fluido menstrual pode ser transportado em condições ideais de temperatura até 24 horas após a coleta. As células tronco mesenquimais podem ser caracterizadas por imunofenotipagem, isoladas, cultivadas e expandidas e, em seguida, criopreservadas. O fluido menstrual contém células tronco mesenquimais viáveis e apropriadas para cultivo<br>INTRODUCTION: Mesenchymal stem cells may renovate different tissues, but techniques to obtain these cells are invasive. Recently, those cells were detected in menstrual blood. OBJECTIVE: Patterning techniques of collection, transportation, characterization, isolation, expansion and cryopreservation of stem cells in menstrual fluid. METHODS: From August 2011 to March 2012 twenty volunteers were selected with regular menstrual cycle without gynecological diseases. They collected menstrual fluid on the most intense flux day to analysis by immunophenotyping and cellular culture. Culture was made in 2 stages until reached semi-confluence of stem cells and these cells were cryopreserved. RESULTS: Average of menstrual fluid volume was 6,90±5,60mL, transportation time was 17,20±5,50h, and total number of cells was 3,95 x106±3,88 x106 witch 76,05%±24,57 were viables. After culture, mesenchymal stem cells increased from 0,14%±0,26 to 96,19%±2,14. After 15 to 21 days of culture in first passage, colonies formed clusters that reached confluence. In second passage, it happens after 3 days of culture and stem cells were cryopreserved. CONCLUSION: Stem cells of menstrual fluid may be easily obtained without invasive methods. Menstrual fluid can be transported in good conditions of temperature up to 24 hours of collection. Mesenchymal stem cells of menstrual fluid may be characterized by immunophenotyping, as well as it is possible to isolated, cultivate and cryopreserved them. Menstrual fluid has viable and proper for culture mesenchymal stem cells

Acute Kidney Disease (CKD) is a major public health problem that affects some 3-7% of patients admitted to the hospital and approximately 25-30% of patients in the intensive care unit. None of the existing therapies are exempt from side effects and kidney physiological functionality is never restored. Transplantation has been reported as the preferred cure for CKD management but organ shortage and risks due to the immunosuppressive therapy makes it far from being the perfect treatment for ESRD. In this study we have focused our attention on finding novel strategies, in vitro and in vivo, to obtain kidney regeneration in case of acute and chronic kidney damage. First we have demonstrated the ability of hAFSCs to survive, proliferate and integrate into the embryonic kidney, while it undergoes organ development, in an in vitro culture system. We observed the presence of hAFSCs within kidney primordial, including tubules and developing nephrons. Thus, hAFSCs seem to have the capacity to undergo the expected mesenchymal to epithelial transition that occurs in normal renal development and are induced to express important early kidney markers such as GDNF, ZO-1 and Claudin. Moreover, hAFSCs do not appear to require prior genetic modification or exogenous production of kidney proteins for their differentiation to occur. This is a very important advantage that hAFSCs have for potential future regenerative or bioengineering application. With the in vivo experiments, we have demonstrated that early direct injection of hAFSCs into the kidney strongly ameliorates ATN injury as reflected by more rapid resolution of tubular structural damage and by normalization of creatinine and BUN levels. In addition, our data show evidence of immunomodulatory and antinflammatory effect of hAFSCs, at an early time point, comparable in magnitude to endogenous cytokine production. Understanding how donor and host cells combine to attenuate tubular damage may lead eventually to the application of hAFSCs for therapeutic purposes in acute kidney diseases. Nonetheless, beside the presence of a small number (1%) of cells with pluripotent characteristics, the composition of the other 99% of Amniotic Fluid cells is diverse, with a great amount of cells exhibiting commitment to a defined germ line or cellular endpoint. There seems to be clear evidence for the existence of progenitor cells in Amniotic Fluid, which can give rise to different cell types of mature organs. By 17 weeks of gestation is notable an increase tissue specific cellular presence and this data may indicate that the choice of the time point for cell selection is fundamental. In addition, we demonstrated in the amniotic fluid, the presence of a renal population with specific traits of commitment. In particular, the presence of podocytes at both undifferentiated and almost mature stages could favour their use for kidney regeneration in vitro and in vivo animal models. The presence and identification of specific renal progenitor cells in the Amniotic Fluid, committed to different compartments of the kidney environment, could represent a valuable new tool for regenerative purposes with regards to the treatment of a broad range of renal diseases. The discovery of renal specific progenitor cells within Amniotic Fluid could bring a breakthrough in the study for novel and more selective approaches in the renal therapy. However, the real pluripotential capability of these progenitors cells, in particular the kidney progenitors presenting more differentiation characteristics, has to be established. Moreover, their potential for survival, proliferation, integration, and differentiation needs to be assessed in in vivo models involving different types of renal damage.<br>L’insufficienza renale terminale ha raggiunto ormai proporzioni epidemiche in tutto il mondo e, tutt’oggi, non sono ancora state trovate terapie sostitutive o rigenerative efficaci a lungo termine. Attualmente la terapia dialitica e il trapianto allogenico rimangono le uniche alternative valide da utilizzare in questi pazienti nonostante se ne conoscano i numerosi limiti e complicanze. Recenti dati epidemiologici, in America e in Europa, mostrano che l’insufficienza renale colpisce circa l’8% della popolazione. [1] L’aumentata domanda di organi, in aggiunta all’insufficiente disponibilita’ di donatori, sta spingendo sempre piu’ i ricercatori di tutto il mondo a sviluppare nuove alternative terapeutiche per la sostituzione dei reni non funzionanti. [2] La creazione di organi bio-artificiali, attraverso l’utilizzo delle tecniche di ingegneria tissutale, ha finora dimostrato grandi difficolta’ specialmente nel riprodurre quegli organi e tessuti la cui struttura e funzione risultino particolarmente complesse, come nel caso dei reni. Storicamente gli ingegneri tissutali che si sono cimentati in questo campo hanno potuto utilizzare esclusivamente linee cellulari adulte dando origine a costrutti bidimensionali caratterizzati da limitata funzione e difficile applicabilita’ in vivo. [3] Nell’ultima dacade le cellule staminali stanno ricevendo sempre maggiore attenzione scientifica grazie al loro crescente impiego nella medicina rigenerativa per la ricostruzione e rigenerazione di tessuti bio-artificiali ed organi. Le cellule Staminali Embrionali (SE), derivate dalla blastocisti, hanno come caratteristiche peculiari il fatto che si replichino ampliamente e che siano capaci di formare aggregati (corpi embrioidi) che possono dar luogo ad una varietà di cellule specializzate come, ad esempio, cellule neurali, cardiache e pancreatiche. [3, 4] Il reclutamento di questo tipo di cellule staminali, tuttavia, comporta la distruzione di embrioni umani creando spinosi problemi etici e morali che portano, in molti Paesi, a vietarne l’utilizzo e il progresso scientifico. Per evitare questo tipo di controversie ricercatori di varie discipline hanno identificato potenziali fonti di cellule staminali alternative. [4, 5] E’ ormai ben noto che in molti tessuti adulti esistono cellule progenitrici con il compito di rigenerare o riparare l'organo a seguito dei fisiologici processi di senescenza o in caso di danno. [6, 7] Ci sono sempre piu’ evidenze che questi progenitori d’organo abbiano caratteristiche di plasticità piu’ elevate di quanto si pensasse originariamente. Parallelamente molti ricercatori credono che la rigenerazione di organi adulti derivi principalmente dalla mobilizzazione di cellule staminali provenienti dal midollo osseo. E’ stato dimostrato che cellule staminali del midollo osseo possono attraversare la barriera endolteliale e dar luogo a differenti linee cellulari differenziate, trasformando cellule circolanti in fegato, cervello, pancreas, pelle, intestino e anche rene. [27, 29] Il liquido amniotico e’stato usato per anni come uno strumento sicuro e valido per la ricerca di malattie genetiche e congenite del feto. Tuttavia, il liquido amniotico contiene un grande numero di cellule progenitrici che posono avere un importante ruolo nelle applicazioni della bioingegneria tissutale. Streubel et al. [8] hanno riportato l’utilizzo di cellule non emopoietiche per la conversione di amniociti in miociti. Recentemente una popolazione di cellule staminali c-Kit+, isolate nel liquido amniotico umano e murino, e’ stata caratterizzata e differenziata in tessuti originati dai tre foglietti embrionali: muscolare, neuronale, adipocitario, epatico, osseo ed endoteliale [9] Nel laboratorio diretto dal dr. R.E. De Filippo, Assistant Professor presso il Childrens Hospital di Los Angeles, abbiamo ampiamente studiato e utilizzato questa nuova popolazione di cellule staminali derivate dal liquido amniotico focalizzando le nostre ricerche sul loro utilizzo nella rigenerazione renale. Abbiamo dimostrato che questa popolazione totipotente di cellule mesenchimali e’ capace di riprodurre alcune tappe essenziali della nefrogenesi dopo essere state iniettate in reni embrionici. Tuttavia, le cellule staminali da liquido amniotico rapresentano meno dell’1% dell’intera popolazione cellulare e forse esistono altri progenitori cellulari, nel liquido stesso, gia’ orientati e piu’ proni alla differenziazione di particolari linee cellulari renali che possano essere utilizzate per gli stessi scopi rigenerativi ma con risultati migliori. Il volume e la composizione del liquido amniotico cambia durante la gravidanza e dall’ottava settimana di gestazione i reni fetali iniziano a produrre liquido che rapidamente aumenta di volume durante il secondo trimestre. [10] Il contatto tra il liquido amniotico e i diversi tessuti fetali sembra giustificare la presenza dei differenti tipi cellulari disciolti nel liquido stesso. La presenza di cellule mature derivanti dai tre foglietti germinali e’ stata gia’ dimostrata in passato come pure la presenza di progenitori cellulari di origine mesenchimale ed emopoietica prima della 12ma settimana gestazionale nell’uomo. [11,12,13] Cellule esprimenti proteine e markers genetici tipici di tessuti diversi come cervello, cuore, e pancreas sono state ritrovate nel liquido amniotico ma ulteriori indagini sono necessarie per completare la caratterizzazione dei diversi tipi cellulari presenti alle diverse eta’ gestazionali. [14, 15, 16] Lo sviluppo renale e’ un complesso processo di attivazione genica, interazioni cellulari ed extracellulari che devono aver luogo secondo un ordine spazio-temporale preciso e nella quantita’ adeguata. Durante l’embriogenesi, il rene metanefrico origina dall’invasione da parte della gemma ureterale, derivata dal dotto epiteliale di Wolffian, nel mesenchima metanefrico. [17] La gemma ureterale inizia la sua arborizzazione all’interno del mesenchima e portera’ alla formazione dell’intero sistema escretore, dall’uretere ai dotti collettori, mentre il mesenchima metanefrico dara’ luogo alle strutture epiteliali costituenti i nefroni (dal tubulo distale alla capsula di Bowman). CD-24 e Caderina 11 sono due markers di superficie che vengono usati per identificare cellule staminali ancora indifferenziate ma presenti nel mesenchima metanefrico prima di ricevere l’induzione da parte della gemma ureterale. [18] In aggiunta, altri markers di superficie che identificano una sottopopolazione di cellule appartenenti al mesenchima metanefrico nei vari stadi dell’induzione verso la nefrogenesi sono stati recentemente descritti in letteratura: Caderine E, PDGFRα, PDGFRβ, e NGFR ad alta affinita’. [19] Cellule Staminali derivate da liquido amniotico e differenziazione renale in vitro e in vivo Negli ultimi sette anni il gruppo di ricerca di cui ho fatto parte per due anni negli Stati Uniti (University of Southern California - Childrens Hospital Los Angeles) sta studiando una popolazione di cellule staminali ricavate da liquido amniotico (Amniotic Fluid Stem Cells, AFSC), umano e murino. Caratterictiche peculiari di questa popolazione cellulare sono: l’espressione di geni e marcatori di superficie comuni a cellule staminali di origine embrionale e mesenchimale; propagazione in vitro senza necessita’ di feeder-layer; mantenimento della lunghezza dei telomeri; capacità di differenziarsi in vitro e in vivo in molti tipi differenti di cellule e tessuti provenienti da tutti e tre i foglietti embrionali. [7] In particolare, negli ultimi 4 anni, il nostro gruppo si e’ concentrato sull’utilizzo di questa particolare popolazione di cellule staminali derivate da liquido amniotico nella rigenerazione renale in vitro e in vivo. [20, 21] Brevemente, siamo stati in grado di dimostrare, basandoci su un sistema in vitro, come le hAFSC abbiano la capacità di differenziarsi in parenchima renale dopo iniezione diretta in reni embrionici (12.5-16 giorni di gestazione) coltivati in vitro fino a 10 giorni. Le cellule staminali da liquido amniotico erano state precedentemente transfettate con il gene codificante una proteina fluorescente verde (GFP) e un secondo gene codificante per il Lac-Z. Mediante queste transfezioni siamo stati in grado di distinguere le cellule iniettate e dopo aver coltivato gli organi, anche a lungo termine (10 giorni), e’ stato possibile dimostrare la loro integrazione ed assimilazione nelle differenti tappe dello sviluppo renale. Analisi istologica dei reni iniettati con le staminali ha rivelato quanto questa popolazione di cellule sia capace di contribuire alle strutture primordiali del rene a partire dalla vescicola renale fino alle ultime fasi della nefrogenesi (tubuli e glomeruli). Mediante RT-PCR abbiamo quindi dimostrato la neoespressione, da parte delle AFSC iniettate, di geni attivi nelle diverse fasi dello sviluppo embrionale del nefrone. [20] Dopo aver dimostrato questa abilità di integrazione nel tessuto renale in via di sviluppo e la compartecipazione a tutte le tappe utili alla formazione del nefrone maturo in vitro, la nostra idea e’ stata quella di procedere all’applicazione in vivo delle cellule staminali da liquido amniotico. L’obiettivo e’ stato quello di dimostrare la loro reale capacità di sopravvivere, replicarsi ed integrarsi attivamente nei reni danneggiati di un modello di topo immunodepresso. Cellule staminali da liquido amniotico di topo (mouse Amniotic Fluid Stem Cells, mAFSC), esprimenti Lac-z e Luciferasi come marcatori, sono quindi state iniettate per via endovenosa (vena della coda) in un modello di topi immunodepressi con tubulonecrosi acuta. Il nostro ultimo obiettivo e’ stato quello di dimostrare se le cellule staminali venissero utilizzate dai reni danneggiati per riparare il danno e quindi fossero in grado di velocizzare la ripresa funzionale dell’organo. I risultati di tali esperimenti hanno dimostrato che le AFSC hanno una buona capacita’, anche in vivo, di integrarsi e partecipare attivamente alla riparazione del danno. Esse hanno iniziato ad esprimere GDNF, un fattore di trascrizione precoce presente nello sviluppo renale e in particolare nella formazione tubulare e glomerulare, e diversi altri markers tubulari quali Acquaporina-2, Agglutinina P, Agglutinina DB. Dagli esperimenti in vivo e’ quindi emerso che la popolazione di cellule staminali totipotenti, derivata da liquido amniotico (hAFSC), e’ capace di differenziarsi in diversi tipi cellulari appartenenti sia a strutture glomerulari (capsula di Bowman) che tubulari (tubulo distale e prossimale) senza dimostrare una chiara specificita’ per una delle due strutture. [9] In accordo con recenti pubblicazioni, abbiamo dimostrato un effetto immuno-modulartorio delle cellule staminali. Lo studio approfondito delle citochine, endogene ed esogene (prodotte dalle hAFSC iniettate), e il loro effetto nel migliorare la porzione infiammatoria del danno renale sono il passo successivo delle nostre ricerche. Un limite potenziale all’utilizzo terapeutico di questa popolazione cellulare totipotente risiede nel fatto che la maggior parte delle malattie renali che portano ad insufficienza renale terminale, colpiscono primariamente le strutture tubulari o quelle glomerulari, ma difficilmente entrambe contemporaneamente. Utilizzando dunque cellule staminali troppo indifferenziate, e quindi totipotenti, si rischierebbe di perdere efficacia terapeutica a causa del fatto che esse riceverebbero troppi segnali contemporaneamente in senso differenziativo e sarebbero indotte a seguire petterns riparativi non mirati e meno efficaci nella riparazione del danno principale. Se infatti avessimo bisogno di trattare selettivamente un danno tubulare piuttosto che uno glomerulare, l’utilizzo di cellule staminali totipotenti non sarebbe cosi’ ottimale come invece l’utilizzo di progenitori tubulo specifici opportunamente espansi ed eventualmente modificati. Questo concetto insieme al fatto che il liquido amniotico e’ composto da differenti popolazioni cellulari ha spinto a considerare la possibilita’ che ci possano essere linee cellulari maggiormente orientate in senso renale (progenitori organo specifici) che possano essere utilizzate in modo piu’ vantaggioso per la rigenerazione di strutture renali specifiche (id. cellule tubulari prossimali o distali, podociti, cellule mesangiali, cellule endoteliali e altro) Caratterizzazione cellulare del liquido amniotico e ricerca di progenitori renali specifici o gia’ parzialmente differenziati L’ultima parte della tesi si e’ concentrata nello studiare ed identificare le varie popolazioni cellulari presenti nel liquido amniotico a diverse settimane di gestazione. I campioni, di eta’ compresa tra le 15 e le 20 settimane di gestazione, sono stati ottenuti tramite amniocentesi, tecnica usata per studiare il cariotipo del feto durante lo sviluppo. Sono stati valutati differenti terreni di coltura, indagando proliferazione e conservazione della morfologia nei campioni ottenuti. L’analisi e la caratterizzazione della popolazione totale presente nel liquido amniotico e’ stata effettuata utilizzando RT-PCR, Real Time PCR e Western Blotting, analizzando l’espressione specifica di geni che sono coinvolti nel mantenimento della pluripotenzialita’, geni che identificano specificatamente i tre foglietti embrionali ed infine geni che identificano progenitori organo-specifici. Sono state inoltre identificate popolazioni specifiche renali, tramite immunoseparazione con biglie magnetiche (MASC). L’espressione di marcatori per i foglietti embrionali endoderma e mesoderma e’ piu’ alta in campioni piu’ giovani rispetto a campioni con tempo di gestazione maggiore mentre, per l’ectoderma, rimane pressoche’ invariata nel tempo. La presenza di cellule pluripotenti e’ costante cosi’ come le cellule staminali mesenchimali mentre le cellule progenitrici ematopoietiche, investigate tramite CD34, fanno la loro comparsa successivamente alle 17 settimane di gestazione. La presenza di progenitori tessuto specifici già “committed” e’ evidente nei campioni di gestazione più avanzata sia per quantitita’ che per specificità dell’organo preso in esame. E’ stata approfondita l’analisi di cellule progenitrici renali, utilizzando un ampio pannello di marcatori che identificano sia la componente tubulare che quella glomerulare del nefrone, struttura fondamentale per la filtrazione renale. I risultati ottenuti confermano la presenza di cellule progenitrici renali dopo le 18 settimane di gestazione. E’ stata identifica e studiata una popolazione esprimente CD24 e Caderin 11 isolata da campioni di liquido amniotico di 18 o piu’ settimane. CD24 e OB-cadehrin sono stati identificati nel topo come co-espressi in vivo nel mesenchima metanefrico. Dal mesenchima metanefrico ha origine il nefrone ed e’ una delle due strutture embrionali fondamentali per lo sviluppo del rene. Da questa popolazione principale sono state ottenute 4 nuove sottopopolazioni che identificano sottocompartimenti del glomerulo, come per esempio le cellule corticali stromogeniche (tramite selezione per la Tyrosin Kinase, TrKA), i podociti (selezionati per la Nefrina), le cellule del mesangio (con selezione positiva per PDGFR Alpha) e le cellule in transizione mesenchima-epitelio (con selezione per la Cadherina-E). Tramite PCR e Real Time PCR e’ stata dimostrata la forte specificita’ di ogni singola linea cellulare. E’ necessario uno studio approfondito che preveda per le AKPC differenziazioni in vitro ed in vivo, utilizzando fattori di crescita nefro-specifici e diversi modelli di danno renale acuto e cronico, in modo tale da confermare la loro possibile completa differenziazione in cellule renali mature. Un approfondimento sul meccanismo d’azione e sulle migliori tempisitiche di somministrazione, infine, sono i punti fondamentali da chiarire per comprendere il meccanismo d’azione delle hAFSC in vivo. Queste ricerche sono una base fondamentale per future applicazioni cliniche in pazienti che soffrono di nefropatie acute e croniche

There is a constant demand for haematopoietic stem cells (HSC) for clinical applications. Amniotic fluid stem (AFS) cells serve as a potential autologous cell source for therapy. Previously murine and sheep AFS have shown to have significant haematopoietic activity after transplantation in immune deficient mice. The haematopoietic potential of Human AFS have never been established in vivo, and its use has been limited by the presence of debris and low cell number at sample collection. My thesis explored the (1) isolation of human amniotic fluid (AF), (2) haematopoietic potential of human AF (CD117/c-Kit+; AFSC) by reconstituting the haematopoietic system of NOD-SCID/IL2rγnull (NSG) mice in vivo and (3) expansion of haematopoietic human AFSC in vitro. Human AF samples (2nd and 3rd trimester, n=110) were collected for the study under an ethically approved project from women undergoing amniocentesis for prenatal diagnosis of congenital disease, or amniodrainage procedures for fetal abnormality. I have employed several strategies to eliminate the large amount of cellular debris from the collected human AF and provide a more homogeneous cell population. Percoll density centrifugation demonstrated a reduction in cell debris and enrichment of the CD117+ population. The haematopoietic potential of human AFSC was explored in vivo. Human AF (2nd and 3rd trimester) and cord blood (CB; control) were selected for CD117 and CD34 respectively. Sorted cells (104 in 200μl PBS) were injected intravenously into sub-lethally irradiated NSG mice (~n=6/group). Human AFSC engrafted the haematopoietic system of NSG mice at levels similar to those achieved with CB-HSC post-primary and secondary transplantation. Importantly, multi-lineage haematopoietic reconstitution was observed at 16 weeks post-primary and secondary transplantation. Moreover, the possibility of expanding haematopoietic progenitors from human AF in vitro was demonstrated with the use of a cytokine-based media and the generation of haematopoietic progenitors by AF derived-induced pluripotent stem cell (AF-iPS) lines. In conclusion, I showed that human AF could be isolated, have long-term multi-lineage haematopoietic potential that is similar to the current “gold-standard” stem cell source for haematopoietic transplantation as well as demonstrates haematopoietic expansion. These findings make human AFSC to be an alternative novel fetal cell source for pre- and post-natal cell or cell-based gene therapy for the treatment of haematological disorders in the future.

The amniotic fluid is a rich source of fetal mesenchymal stem cells with broad differentiation capacity. Human amniotic fluid stem cells (AFSCs) have a high expansion potential, fast growth kinetics and harbour therapeutic potential to treat a variety of conditions. Moreover, they are easily isolated at mid-trimester or at delivery and can be used without ethical restrictions. Osteogenesis imperfecta (OI) is a genetic disease characterised by bone fragility, due to production of abnormal collagen type I. In this study, human AFSCs were transplanted into immunocompetent oim mice (OI mouse model, n=28) at birth. Bones were harvested after 8 weeks and analysed for mechanical properties, micro-structure, engraftment of donor cells and gene expression. Non-transplanted oim and wild-type mice were used as controls. Human AFSC injection decreased bone fracture rate and increased bone strength. Donor cells migrated to the bones, engrafted into sites of active bone formation and appeared to differentiate into osteoblasts, producing normal collagen. Moreover, transplantation improved the microarchitecture of the bones, although bone volume remained unaffected. Transplantation also promoted endogenous osteogenesis, with mouse genes involved in osteoblast differentiation and skeletal development significantly up-regulated, compared to non-transplanted mice. As a side project, a protocol for the isolation and differentiation of human fetal osteoblasts from the calvaria was optimised and a biobank of 24 samples, from gestational ages ranging from 9 to 21 weeks post-conception, was established. The data presented in this thesis indicate that human AFSCs are a promising source for cell therapy in OI. Donor cells may exert their therapeutic effects both by normalising the ECM and by influencing the maturation of resident osteoblasts. Ongoing work is focused on the further understanding of the mechanism of action of donor cells, using co-culture experiments with human AFSCs and human fetal osteoblasts.

We have recently characterized two distinct populations of Satellite Cells (SCs), defined as Low Proliferative Clones (LPC) and High Proliferative Clones (HPC), that differ for proliferation, egenerative potential and mitochondrial coupling efficiency. In here, we have deep investigated their cell biology and characterized features that remark their intrinsic differences retrievable also at the initial phases of their cloning. LPC and HPC can indeed be istinguished for characteristic mitochondrial membrane potential (ΔΨm) just after isolation from their parental fibre. This is merged by mitochondrial redox state measured via NAD+/NADH analysis- and alternative respiratory CO2 production in cloned cells, which are accountable for metabolic differences reflected by alternative expression of the glycolytic enzyme Pfkfb3. In addition also mitochondrial Ca2+ handling and the sensitivity to apoptosis triggered via the intrinsic pathway are modified as well as the size of the mitochondrial network. In conclusion, we were able to determine which clone represents the suitable stem cell within the SCs population. These further experimental observations report novel physiological features in the cell biology of SCs populations before and after cloning, highlighting an intrinsic heterogeneity on which the stemness of the satellite cell is likely to depend. In the second part of my work we have also investigated their potential to trans-differentiate into smooth muscle cells. Enteric Nervous System normally interacts with muscle cells to control the peristaltic and secretory activity of the gut wall. Incomplete gut colonization by neural crest cells causes Hirschsprung’s disease, characterized by aganglionosis of the distal bowel. Multipotent, self-renewing enteric precursor neurosphere-like bodies (NLBs) -capable of generating neurons and glia derived from the neural crest- can be isolated from the gut of mice, rats, and human and they are able to colonize the gut after transplantation. Our aim is to understand the relationship between satellite cells-derived muscle precursor cells (MPCs) and NLBs using an in vitro co-culture model: this will be useful in perspective of a tissue engineering approach for bowel regeneration and skeletal muscle. Our records highlighted that NLBs were able to form new myotubes in presence of MPCs. Co-cultures in myogenic medium showed a remarkable improvement of MPCs ifferentiation by NLBs, promoting the formation of sarcomeric striatures onto myotubes and increasing the desmin expression of MPCs. On the other side, using neurogenic medium MPCs-NLBs showed a neural-like phenotype. As future perspectives, we need to understand the relationship between MPCs and NLBs and if the synapses are involved in this process; to verify if the seeding on a biocompatible polymer influences the behaviour of neural cells; and we must confirm these data with an in vivo skeletal and smooth muscle differentiation. We have finally explored the possibility of deriving smooth muscle cells from a different source, taking in consideration the difficulties related to the expansion of both skeletal and smooth muscle progenitors. Therefore, we aim to derive functional smooth muscle cells (SMCs) from non-muscle cells, such as human Amniotic Fluid Stem (hAFSC) cells. hAFSC were transduced using vector encoding ZsGreen under the αSMA promoter. SMhAFSC expressed significantly higher level of smooth muscle genes (such as αSMA, desmin, calponin and smoothelin expression) after selective culture condition. These features were confirmed by immunofluorescence, demonstrating a single lineage commitment; TEM established increased intermediate filaments, dense bodies and glycogen deposits in SMhAFSC, similar pattern compared to SMCs; and sequential imaging analyses demonstrated that SMhAFSC have a higher contractile potential than hAFSC. Consecutive single cell sampling showed the presence of voltagedependent calcium activated potassium channels on differentiated SMhAFSC and showed a higher production of carbon dioxide. In conclusion, we were able to generate to functional SMCs starting from a non-muscle precursor; secondly the transduction process may represent a valuable tool to select SM committed population. This step may eventually overcome the well-known problem of expanding SM progenitors, making these cells amenable to tissue engineering.<br>Il nostro gruppo ha recentemente caratterizzato due distinte popolazioni di cellule satelliti, classificate come cloni a bassa proliferazione (LPC) e ad alta proliferazione (HPC), che si differenziano in termini di proliferazione, potenziale rigenerativo e metabolismo mitocondriale. Nel mio lavoro di dottorato, abbiamo valutato e caratterizzato la loro biologia cellulare con particolare attenzione a quelle differenze intrinseche presenti anche prima della loro clonazione. Infatti, ambo le tipologie clonali possono essere distinte mediante il potenziale di membrana mitocondriale (ΔΨm) subito dopo l’isolamento dalla fibra. Questo dato è in accordo con lo stato ossido riduttivo mitocondriale misurato tramite NAD+/NADH e la quantificazione della produzione di CO2. Questi risultati sono responsabili delle differenze metaboliche e possono essere spiegati dalla diversa espressione dell’enzima glicolitico Pfkfb3. Inoltre la concentrazione mitocondriale del Ca2+ e la sensibilità all’apoptosi sono modificate così come la dimensione della rete mitocondriale. In conclusione, siamo stati in grado di determinare quale clone rappresenta la cellula staminale all’interno della popolazione di cellule satelliti. Queste nuove osservazioni sperimentali rivelano caratteristiche fisiologiche della biologia delle popolazioni delle cellule satelliti prima e dopo la clonazione, mettendo in luce un’eterogeneità intrinseca della cellula satellite. Nella seconda parte della mia tesi abbiamo esplorato la possibilità che le cellule satelliti possano, se opportunamente stimolate, trans-differenziarsi in cellule muscolari lisce. Il sistema nervoso enterico normalmente interagisce con le cellule muscolari per controllare l’attività peristaltica e secretoria della parete intestinale. L’incompleta colonizzazione dell’intestino da parte delle cellule della cresta neurale provoca la malattia di Hirschsprung, caratterizzata da aganglionosi del colon distale. Le neurosfere (NLBs), precursori enterici in grado di auto-rinnovarsi, possono generare neuroni e glia; essere isolate dall’intestino di topi, ratti e umani e sono in grado di colonizzare l'intestino dopo il trapianto. Il nostro obiettivo è di capire la relazione tra i precursori di cellule satelliti (MPCs) e NLBs utilizzando un modello in vitro di co-coltura: questo sarà utile in prospettiva di un approccio di ingegneria tissutale per la rigenerazione intestinale e muscolo scheletrico. I nostri dati hanno evidenziato che NLBs, in presenza di MPCs, sono in grado di formare nuovi miotubi. L’uso di terreni di coltura miogenici ha evidenziato un notevole aumento della differenziazione in senso muscolare, promuovendo la formazione di striature ed aumentando l’espressione di desmina. Dall’altra parte, l’utilizzo di terreni di coltura neurogenici ha mostrato un fenotipo simil neurale. Come prospettive future, dobbiamo comprendere ulteriormente la relazione tra MPCs e NLBs e se le sinapsi sono coinvolte in questo processo; si deve verificare se un loro utilizzo su polimeri biocompatibili ne possa influenzare il comportamento, ed infine è necessaria una conferma dei suddetti dati tramite un’analisi di differenziazione in vivo in muscolo scheletrico e liscio. Nella terza ed ultima fase del mio lavoro, ci siamo focalizzati ad esplorare la possibilità che cellule non-muscolari possano, se opportunamente stimolate, differenziare in senso muscolare liscio. Il nostro obiettivo è stato quello di ottenere cellule muscolari lisce (SMCs) partendo da cellule staminali del fluido amniotico umano (hAFSC). hAFSC sono state trasdotte utilizzando un virus codificante per ZsGreen sotto il promotore αSMA. SMhAFSC così ottenute hanno evidenziate un alto livello d’espressione dei geni del muscolo liscio (come αSMA, desmina, calponina e smoothelin). Queste caratteristiche sono state confermate da molteplici analisi: di immunofluorescenza, dimostrando la positività a marcatori specifici per il muscolo liscio; microscopia a trasmissione elettronica (TEM), dove si verificava l’aumento della presenza di filamenti intermedi, di corpi densi e depositi di glicogeno, modello simile rispetto alle SMCs. Analisi in timelapse di SMhAFSC hanno dimostrato che queste possiedono un potenziale contrattile superiore rispetto hAFSC e studi su singola cellula hanno evidenziato la presenza di canali calcio voltaggio-dipendenti attivati da potassio solamente su SMhAFSC. In conclusione, siamo stati in grado di generare di cellule muscolari lisce funzionali da un precursore nonmuscolare ed in secondo luogo il processo di trasduzione può rappresentare un valido strumento per distinguere e selezionare differenti popolazioni. Questa fase può eventualmente superare il ben noto problema dell’espansione di progenitori di cellule muscolari lisce, rendendo queste cellule suscettibili per approcci d’ingegneria tessutale.

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.

Introduction: Stem cell biology has received much interest because of its potential in both therapeutic application and in vitro modeling of diseases. In particular embryonic stem cells have good proliferative and differentiative abilities, but their use is still associated to ethical concerns and problems related to their teratogenic potential. Adult stem cells have also been described to be pluripotent both in vitro and in vivo. However, their use is limited because they are difficult to isolate and expand, particularly in a clinical setting. In this scenario, it would be advantageous to obtain a cell population with high selfrenewal and differentiation capacities, without ethical problems. In 2007 our group described that amniotic fluid stem (AFS) cells could be derived selecting amniocytes using c-Kit antibody. AFS cells have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Aim: This work aiming at characterize the myogenic potential of mouse AFS cells using a mouse model of spinal muscular atrophy and in particular at analyzing their ability to differentiate into satellite cells and colonize the muscle stem cell niche. Materials and Methods: Mouse AFS cells were obtained by amniocentesis and selected for the marker c-Kit with immunomagnetic beads. Freshly isolated AFS cells were analyzed for the expression of different markers (CD90, CD45, CD44, CD34, CD31, Flk1, SCA1, CD105) by flow cytometry and the expression of Oct4, Sox2, c-Myc, Klf4 and Sca-1 by qRT-PCR at different embryonic stages. For the treatment of HSA-Cre, SmnF7/F7 mutant mice, GFP+ AFS cells were injected via the tail vein and animals were sacrificed one and fifteen months after transplantation. Clinical aspects were observed and analyzed after transplantation to evaluate AFS cells’ effects. Several muscles were stained with hematoxylin and eosin, Masson’s trichrome and analyzed by immunofluorescence with anti-GFP and anti-dystrophin antibodies. To demonstrate the ability of AFS cells to replenish the muscle niche, staining for satellite cell markers and secondary transplantation were performed. The myogenic potential of AFS cells was also evaluated with transplantation after in vitro expansion. Results: Mouse AFS cell number changes during the course of gestation. At E12.5 these cells express hematopoietic markers (CD45, CD34, SCA1), mesenchymal markers (CD90, CD105) together with Flk1, CD31 and CD44. Gene expression analysis showed that mouse AFS cells express at low levels Oct4 and Sox2 and at high levels c-Myc and Klf4, whereas they are negative for the expression of myogenic genes. Mild muscular mutant HSA-Cre, SmnF7/F7 mice die at the age of 10 months and show evident clinical complications such as kyphosis and muscle shrinkage. After transplantation with GFP+ AFS or bone marrow (BM) cells mice survival rate increased by 75% and 50% respectively. Animals treated with AFS cells recovered more than 75% of force compared to the untreated. One month after transplantation, muscles obtained from AFS-treated mice displayed 37% of GFP+ fibers, with very low number of regenerating myofibers (<1%) and normal dystrophin expression. Fifteen months after transplantation BM-treated mice displayed a high number of central nucleated fibers and consistent infiltration of interstitial tissue and no GFP+ myofibers, while AFS-treated mice had a normalized phenotype, close to the same age WT mice, and 58% of the myofibers were GFP+. Similar results were obtained with transplantation of mouse AFS cells expanded in culture. Discussion: Mouse AFS cells are a heterogeneous population, and their phenotype changes during the course of gestation. At E12.5 they express mesenchymal, hematopoietic and endothelial markers, but most importantly don not express myogenic factors, indicating that no myogenic progenitor cells are present in this stem cell population. When injected in a muscular mutant mouse model, AFS cells showed a myogenic potential, even after long-term transplantation, suggesting an interesting therapeutic potential. They indeed were able to differentiate into satellite cells localizing in the muscle stem cell niche and expressing Pax7, a7integrin and SM/c-2.6, exclusively markers of satellite cell population. Moreover, AFS cells could contribute to the formation of new myofibers even after in vitro expansion.<br>Introduzione: Negli ultimi anni lo studio delle cellule staminali ha suscitato molto interesse, sia per il grande potenziale di queste cellule nelle terapie e applicazioni cliniche, sia come modello di studio in vitro per diversi tipi di malattie. In particolare, le cellule staminali embrionali hanno una elevata capacità proliferativa e di differenziazione, ma il loro utilizzo è ancora associato a problematiche etiche. Anche le cellule staminali adulte possiedono grandi potenzialità differenziative sia in vitro che in vivo, tuttavia il loro utilizzo è limitato in quanto difficili da isolare ed espandere, soprattutto in ambito clinico. In questo scenario sarebbe vantaggioso poter ottenere una popolazione di cellule con elevata capacità di proliferazione e differenziazione, senza dover affrontare però problemi di tipo etico. Nel 2007 il nostro gruppo ha isolato una popolazione di cellule staminali dal liquido amniotico (cellule AFS), utilizzando come marcatore il recettore c-Kit. Queste cellule hanno capacità clonogenica e possono essere dirette a differenziare in una vasta gamma di tipi cellulari appartenenti a tutti e tre i foglietti germinativi. Obiettivo: Questo lavoro mira a caratterizzare il potenziale miogenico delle cellule staminali del liquido amniotico di topo utilizzando un modello murino di atrofia spinale muscolare. In particolare è volto ad analizzare la capacità delle cellule AFS di dare origine a cellule staminali muscolari e colonizzare la nicchia staminale del muscolo scheletrico. Materiali e Metodi: Le cellule AFS sono state ottenute mediante amniocentesi e selezionate per la positività al marcatore c-kit con metodo immmunomagnetico. Appena isolate le cellule AFS sono state analizzate per l'espressione di diversi marcatori (CD90, CD45, CD44, CD34, CD31, Flk1, SCA1, CD105) tramite citometria a flusso; inoltre, attraverso qRT-PCR è stata analizzata l'espressione di Oct4, Sox2, c-Myc, Klf4 e Sca-1 delle cellule AFS isolate a diversi stadi embrionali. Per la terapia di topi transgenici HSA-Cre, SmnF7/F7, le cellule AFS GFP+ sono state iniettate per via sistemica attraverso la vena caudale; gli animali sono stati poi sacrificati a uno e a quindici mesi dopo il trapianto. Sono stati osservati e analizzati alcuni parametri clinici per valutare l’effetto del trapianto cellulare. Diversi muscoli sono stati raccolti ed analizzati con ematossilina e eosina, tricromica di Masson e mediante immunofluorescenza con anticorpi anti-GFP e anti-distrofina. Per dimostrare la capacità delle cellule AFS di colonizzare la nicchia staminale del muscolo, sono state eseguite delle immunofluorescenze per i marcatori specifici delle cellule satelliti e sono stati eseguiti dei trapianti secondari. Il potenziale miogenico delle cellule AFS è stato valutato anche con trapianto dopo espansione in vitro. Risultati: Il numero medio di cellule AFS presenti nel liquido amniotico varia nel corso della gestazione murina; all’età di 12.5 giorni queste cellule sono circa l’1% del totale ed esprimono marcatori ematopoietici (CD45, CD34, SCA1), marcatori mesenchimali (CD90, CD105) unitamente a Flk1, CD31 e CD44. L’analisi di espressione genica ha dimostrato che le cellule AFS esprimono a bassi livelli Oct4 e Sox2 e alti livelli di c-Myc e Klf4, mentre, nonostante la composizione mista di questa popolazione, non è stata rilevata espressione di marcatori o fattori di trascrizione tipici dei precursori muscolari. I topi HSA-Cre, SmnF7/F7 mediamente muoiono all'età di 10 mesi e durante il corso della loro vita mostrano evidenti complicazioni cliniche come una pronunciata cifosi e atrofia a livello muscolare. Dopo il trapianto con cellule AFS GFP+ o con cellule del midollo osseo, il tasso di sopravvivenza di questi animali aumenta rispettivamente del 75% e 50%. Gli animali trattati con cellule AFS hanno recuperato più del 75% della forza rispetto agli animali non trattati. Un mese dopo il trapianto, i muscoli di topi trattati con cellule AFS presentano il 37% di fibre GFP+, un numero molto basso di miofibre rigeneranti (< 1%) ed una normale espressione di distrofina. Quindici mesi dopo il trapianto, gli animali trattati con cellule del midollo osseo mostrano un elevato numero di fibre centro nucleate, un’importante infiltrazione di tessuto interstiziale e nessuna miofibra GFP+, mentre i topi trattati con cellule AFS hanno un fenotipo molto simile a quello di topi sani della stessa età, e il 58% delle miofibre è GFP+. Risultati simili sono stati ottenuti trattando lo stesso modello animale con cellule AFS dopo espansione in cultura. Discussione: Le cellule AFS isolate dal liquido amniotico di topo sono una popolazione eterogenea; queste cellule esprimono marcatori mesenchimali, ematopoietici e marcatori endoteliali. Va evidenziato che, nonostante la composizione mista di questa popolazione staminale, non esistono precursori muscolari al suo interno, e quindi qualunque differenziamento in senso muscolare di queste cellule è dovuto ad una differenziazione delle cellule AFS e non ad una maturazione di cellule già pre-commited. Quando vengono iniettate in un modello di atrofia muscolare, le cellule AFS mostrano un grande potenziale miogenico, anche a lungo termine, dimostrandosi una interessante fonte cellulare per scopi terapeutici. Queste cellule infatti sono state in grado di differenziare in cellule satelliti localizzandosi nella nicchia delle cellule staminali muscolari ed esprimendo Pax7, a7integrina e SM/c-2.6, tutti marcatori esclusivi delle cellule satelliti. Inoltre, le cellule AFS possono contribuire alla formazione di nuove miofibre anche dopo espansione in cultura, aumentando così lo spettro di possibili applicazioni terapeutiche.

Abstract Background Mesenchymal stem cells (MSC) are non-hematopoietic multipotent stromal cells that can differentiate into lineages such as adipocytes, osteocytes and chondrocytes. MSC are immune privileged and also possess immunosuppressive properties, which in combination with their differentiative properties makes them excellent candidates for tissue engineering, an alternative treatment solution for of congenital malformations. This study will investigate mesenchymal stem cells from amniotic fluid and umbilical cords to evaluate which tissue that is superior for tissue engineering. Methods and Results Mesenchymal stem cells were isolated from amniotic fluid (afMSC) obtained at routinely performed amniocenteses and from umbilical cords (ucMSC) collected at births from elective caesarean sections. afMSC were cultured in Dulbecco’s modified Eagle medium-low glucose (DMEM) and ucMSC isolated with 3 different protocols were cultured in two different media; modified Eagle medium Alpha (MEMα) and DMEM. After expansion, the cell populations were characterized in regards to their phenotype, immunological properties, proliferative capacity and in vitro differentiation abilities. Mixed lymphocytes culture (MLC) showed that the af and ucMSC were immune privileged and also possessed immunosuppressant properties. Furthermore, the cells cultured in MEMα suppressed the immune response to a greater extent than cells cultured in DMEM. MSC from both sources showed varied differentiation potential towards the osteogenic and adipogenic lineages, but overall with low efficiency. All cells were positive for CD105, CD44, CD73 and HLA I and negative for CD45, CD31, CD14, CD80, HLA-DR and HLA II. MSC from uc were negative for CD34 and positive for CD90, whereas afMSC were intermediately positive for CD34 and CD90. The cells were cultured for 10 passages to investigate their proliferative capacity and their population doubling times were calculated. The average doubling time for afMSC was 49.3 ± 12,7 hours at passages 1 to 5, 42.3 ± 7.5 hours for ucMSC. Conclusions Based on this data we will in the forthcoming studies isolate cells from amitotic fluid using positive selection for various markers and culture the cells in MEMα. With these changes we hope to obtain a potent homogenous cell population that can be used in the treatment of congenital malformations.

Amniotic fluid stem cells (AFSCs) offer therapeutic potential for prenatal and neonatal diseases based on their unique features. From the development of embryos, AFSCs represent a category between embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), with AFSCs more primitive than MSCs. Our lab has previously established that AFSCs can be reprogrammed to regain functional pluripotency with valproic acid (VPA). However, detailed mechanisms are still unknown. Here, our results showed that Wnt signalling was downregulated in the initial stage and upregulated with VPA treatment; whereas mesenchymal-to-epithelial transition (MET) was not observed in the process. Additionally, our previous results demonstrated that AFSCs maintained in ESC conditions shared 82% transcriptome similarity with ESCs. In the second part of this study, we revealed that features of AFSCs including marker expression and differentiation ability were sustained better in ESC conditions. Regarding osteogenesis, enhanced osteogenic ability was found in AFSCs maintained in ESC conditions due to a TGF-beta/CD73-dependent signalling pathway. Moreover, in addition to possessing the same tri-lineage differentiation capability as MSCs, AFSCs can also be induced to express cardiac markers, but contractile cells have not been obtained yet. As features of AFSCs are better preserved in ESC conditions, a Wnt-dependent cardiomyocyte differentiation protocol for pluripotent stem cells is examined on AFSCs in the last part of this study. Our results showed that, with the Wnt-dependent protocol, cardiac markers were induced but spontaneously contractile cells were not observed. Taken together, our findings show that (1) Wnt signalling may play a role in VPA-induced reprogramming, (2) AFSCs maintained in ESC conditions can better maintain stem cell features, especially osteogenic ability through a TGF-beta/CD73 pathway, (3) With a Wnt-dependent protocol, AFSCs can be induced to express cardiac markers but not to become contractile cells.

Amniotic fluid stem (AFS) cells can be expanded without feeder layers and can differentiate into mesenchymal and haematopoietic lineages. Long term engraftment has been difficult to achieve after prenatal stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained from amniotic fluid (AF) with minimal risk. My thesis aims to define the potential of human, sheep and mouse AFS cells as an autologous stem cell source for prenatal cell/gene therapy. Using pregnant sheep, I explored using AF mesenchymal stem cells (AFMSCs) and CD34+ cells for autologous in utero therapy. AF was collected under ultrasound-guided amniocentesis in early gestation. Those cells were transduced with enhanced green-fluorescent-protein (GFP) using lentivirus vector. After expansion, transduced AFMSCs were injected into peritoneal cavity of each donor fetal sheep. Widespread transgenic GFP expression was detected in fetal tissue. For looking into haematopoietic potential, I transplanted autologous fresh and frozen CD34+AFS, and bone marrow cells into immunocompromised mice. Sheep CD34+AFS cells formed colonies, and were positive for CD45, but negative for CD14/CD31/CD4/ CD58. Flow cytometric analysis at 3 months showed GFP positive cells in all haematopoietic organs. To prove congenic transplantation is better than allogeneic, I collected AF from YFP+/C57BL/6 mice at E13. CKit+/Lin- cells were injected into peritoneal cavity of every mouse fetus. Peripheral blood engraftment was significantly higher in mice transplanted with congenic, versus allogeneic cells, as was liver and spleen engraftment. Finally, looking ahead to clinical translation, human AF cells could be cultured, transduced, sorted and expanded in vitro by using conditional medium in adherent plates. Xenogeneic transplantation of human Ckit+ AFS cells into fetal mice also showed minimal engraftment in peripheral blood. In conclusion, AF derived stem cells are an important source of autologous cells that could have prenatal therapeutic value in cell or cell-based gene therapy in the future.

Brain injury, either surgically induced or as a result of trauma or stroke, is one of the leading causes of death and disability worldwide. Since transplantable stem cell sources are showing a great deal of promise and are actively being pursued to provide neuroprotection post-injury, in this body of work, we set out to characterize and examine the therapeutic potential of amniotic fluid derived (AF) cells as a potential cell source for cell-based therapies in mediating neuroprotection post-injury. Despite their heterogeneity, we found that AF cells are mainly epithelial in origin and express various genes involved in stem cell maintenance and neural commitment. A very small subset of AF cells also express pluripotency markers OCT4a, SOX2 and NANOG, which can be enriched for by single cell cloning. SOX2 positive clones have the capacity to give rise to a neuronal phenotype, in neural induction conditions, which can be used to examine the neural differentiation capabilities of AF cells. Subsequently, we examined the ability of AF cells to mediate a neuroprotective effect in a surgically induced brain injury model through gap junctional-mediated direct cell-cell communication and as a vehicle for GDNF delivery post-injury. AF cells express high levels of CX43 and are able to establish functional gap junctional intercellular communication (GJIC) with cortical astrocytes. We report an induction of Cx43 expression in astrocytes following injury and demonstrate, for the first time, CX43 expression at the interface between implanted AF cells and host astrocytes. In an effort to boost host endogenous neuroprotective mechanisms post-injury, via neurotrophic factor delivery, we engineered AF cells to secrete GDNF (AF-GDNF). GDNF pre-treatment significantly increased AF cell and cortical neuron survival rates following exposure to hydrogen peroxide. AF-GDNF cells, seeded on polyglycolic acid (PGA) scaffolds, survived longer in serum-free conditions and continued to secrete GDNF post-implantation activating the MAPK/ERK signaling pathway in host neural cells in the peri-lesion area. Despite some promising trends, we did not observe significant behavioural improvements following AF-GDNF/PGA implantation nor reduced lesion volume during the 7 day time-frame. In conclusion, through GJIC with cortical astrocytes and delivery of exogenous neurotrophic factors, AF cells hold great promise in mediating neuroprotection post-injury.

Introduction: Stem cells are defined by their ability to proliferate for a long period of time, a property known as ‘self-renewal’, and to give rise to differentiated cells. Stem cells can be distinguished into totipotent, pluripotent, multipotent, oligopotent and unipotent. They can also be classified into embryonic, adult and fetal stem cells. Embryonic stem (ES) cells are obtained from inner cell mass (ICM) of blastocyst and are puripotent. Primordial germ cells (PGC) in the embryo give rise to gametes but they are not pluripotent, albeit they express Oct4, Nanog and Sox2. They can be reprogrammed in vitro, becoming pluripotent embryonic germ (EG) cells. Amniotic fluid stem (AFS) cells are fetal stem cells that can be isolated from the amniotic fluid (AF) by the expression of the marker c-kit, both in human and mouse, but their origin is unknown. Human AFS are multipotent in vitro, while both human and mouse AFS have hematopoitic potential, in vitro and in vivo. Recently it has been demonstrated that human AFS from first and second thrimester can be reprogrammend into pluripotent cells in vitro, after supplementation with Valproic Acid (VPA). Cells from the AF have also been used to obtain induced pluripotent stem (iPS) cells. For all these reasons AFS cells seems to be a promising sources of cells for regenerative medicine. Spinal muscular atrophy (SMA) is an autosomal recessive disease, caused by an homozygous deletion or mutation of the motor neuron 1 (SMN1) gene. Bone marrow (BM) transplantation in a murine model of SMA attenuates the myopathic phenotype without a full recovery and without long-term therapeutic effects. Aims of the thesis: Characterisation of fresh mouse AFS cells, evaluation of their myogenic potential into a model of SMA (HSA-Cre, SmnF7/F7 mouse) investigation of their putative PGC origin and induction to pluripotency through a non-viral method (PiggyBac, PB). Materials and Methods: Mouse AFS cells were obtained by amniocentesis and selected as c-kit+ cells with magnetic beads. Freshly isolated-AFS cells were analized for the expression of different markers (CD90, CD45, CD44, CD34, CD31, Flk1, Sca1, CD105) by flow citometry and the expression of Oct4, Sox2, c-Myc, Klf4 and Sca-1 by qRT-PCR at different embryonic stages. Hematopoitic potential was evaluated in vitro, while the teratoma assay was performed in Rag2-/-γc-/- mice. For the reprogramming into EG cells cells were seeded into a feeder layer of mitotically inactivated STO or Sl4-m220, in a PGC medium supplemented with LIF and basic fibroblast growth factor (bFGF) and in N2B27 2iLIF medium. For the treatment of HSA-Cre, SmnF7/F7 mice, GFP+ cells were injected via the tail vain and sacrificed one month after transplantation. Tibialis Anteriour (TA) muscles were stained with hematoxylin and eosin, Masson’s trichrome and analized by immunofluorescence for positivity for GFP/dystrophin. The experiments for the origin of AFS have been conducted using two mouse models: Oct4-GFP and TNAP-Cre. For the induction to pluripotency cells were obtanied from Oct4-GFP positive embryos, and transfected with the PB-TET transposon plasmid containing four genes (Oct-4, Sox-2, c-Myc and Klf4) under the transcriptional control of the tetO2 tetracycline/doxycycline inducible promoter. The expression of pluripotency gene was induced with doxycycline. iPS cells obtained were tested for the expression of Nanog, SSEA-1 and for positivity to alkaline phosphatase. Results: Mouse AFS number chaged during the course of gestation. These cells expressed hematopoietic markers (CD45, CD34, Sca1), mesenchymal markers (CD90, CD105) together with Flk1, CD31 and CD44. On the basis of c-kit expression two populations were defined: c-kithigh and c-kitlow which showed differential expression of the aforementioned markers. c-kitlow are the more abundant, but during the course of gestation they decreases in numbers while the number of c-kithigh cells increases. Both populations had hematopoietic potential vitro. Gene expression analysis showed that mouse AFS cells expressed at low levels Oct4 and Sox2 and high levels c-Myc and Klf4, and their expression changed during the course of gestation. Single cell PCR showed that at E13.5 there 5% of cells co-expressed Oct4, Sox2 and Klf4. Mouse AFS cells didn't form teratoma. In the cell therapy experiments HSA-Cre, SmnF7/F7 control mice died at the age of 10 months, while mice treated with GFP+ AFS or bone marrow (BM) cells had a survival rates increased by 75% and 50% respectively. HSA-Cre, SmnF7/F7 mice treated with AFS cells recovered more than 75% of force compared to the untreated animals. One month after transplantation, muscles from AFS-treated mice displayed very low number of regenerating myofibers (<1%) and normal dystrophin expression; moreover, 37.86% (± 9.48%) of the fibers were GFP+. 15 months after transplantation BM-treated mice displayed a high number of central nucleated fibers and consistent infiltration of interstitial tissue and no GFP+ myofibers, while AFS-treated mice had a mild-phenotype, close to wild-type mice, and 58.00% (± 2.43%) of the myofibers were GFP+. Similar results were obtained with HSA-Cre, SmnF7/F7 treated with mouse AFS cells expanded in culture. To evaluate if mouse AFS cells were PGC cells, they were cultivated following the protocol established to obtain pluripotent EG cells from PGC cells. Two different culture protocols were used, but no EG cells were obtained. AFS cells isolated from Oct4-GFP fetuses at different embryonic stages showed no presence of Oct4+ cells. The TNAP-Cre line resulted to be unspecific. iPS clones obtained transfecting mouse AFS cells were doxycycline indipendent, they expressed Oct4, they were positive for Nanog and SSEA1, and for the alkaline phosphatase. Discussion: Mouse AFS cells are an heterogenous population, and their phenotype changed during the course of gestation. They expressed mesenchymal, hematopietic and endothelial markers. The two populations (c-kithigh and c-kitlow) should be tested in vivo to asses their differentiative potential. Gene expression analysis at population and single cells level confirmed the heterogeinity of mouse AFS cells. AFS showed a myogenic potential, even after long-term transplantion, suggesting an interesting therapeutic potential of these cells. AFS could contribute to the formation of new myofibers by fusing with existing ones or after integration within the stem cell niche of the muscle. The study of their origin suggested that mouse AFS cells aren't PGC. However it is important to remind that the Oct4-GFP mouse is not a lineage-tracking model; therefore more experiments are needed to confirm these results and to find the origin of these cells. iPS cells are a promising research tool to obtain a model of several diseases or as a source of cells for therapeutic approaches. Here it has been shown that the PB system is a suitable method for the reprogramming of mouse AFS cells. These are only preliminary results and more experiments will be necessary to complete the characterisation of these cells.<br>Introduzione: Le cellule staminali hanno la capacità di dare orgine ad una progenie di cellule mature mantendo la capacità di autorinnovamento. Possono essere distinte sulla base delle loro potenzialità in totipotenti, pluripotenti, multipotenti, oligopotenti e unipotenti. Possono anche essere distinte in cellule staminali embrionali, fetali e adulte. Le cellule staminali embrionali, ottenute dalla massa cellulare interna della blastocisti, sono pluripotenti. Nell'embrione le cellule primordiali germinali danno origine ai gameti, ed esprimono i marcatori di pluripotenza (Oct4, Nanog, Sox2), ma non sono pluripotenti. Esse possono essere riprogrammate in vitro, diventando così cellule germinali embrionali. Tra le cellule staminali fetali ci sono le cellule staminali del liquido amniotico (AFS). Queste cellule sono isolate dal liquido amniotico per la positività al marcatore c-kit e sono presenti sia nell'uomo che nel topo, anche se la loro origine embrionale non è nota. Le cellule AFS umane sono multipotenti in vitro; le cellule AFS umane e murine hanno uno specifico potenziale ematopoietico, in vivo e in vitro. Recentemente è stato dimostrato che le cellule AFS umane ottenute dal primo e dal secondo trimestre di gravidanza, possono essere riprogrammate in vitro in cellule pluripotenti, a seguito dell'aggiunta di acido valproico. Inoltre, le cellule del liquido amniotico sono state anche utilizzate da diversi gruppi di ricerca per ottenere cellule staminali indotte alla pluripotenza (iPS). Per questi motivi, le AFS rappresentano una sorgente innovativa di cellule per approcci di medicina rigenerativa. L'atrofia spinale muscolare (SMA) è una malattia autosomica recessiva, causata della delezione o mutazione omozigote del gene della sopravvivvenza del motoneurone 1 (SMN1). Il trapianto di midollo osseo in un modello murino di SMA attenua il fenotipo miopatico, tuttavia non lo recupera totalmente e non mostra alcun effetto benefico a lungo termine. Scopo della tesi: Gli scopi di questa tesi consistono nella caratterizazzione delle cellule murine AFS isolate a fresco, nella valutaione del loro potenziale miogenico dopo il trapianto in animali HSA-Cre, SmnF7/F7, nello studio della loro origine embrionale e nell'induzione alla pluripotenza usando un metodo non virale (PiggyBac, PB). Materiali e Metodi: Le cellule murine AFS sono state ottenute attraverso amniocentesi e successiva immunoselezione per il marcatore c-kit mediante biglie magnetiche. Le cellule AFS sono state analizzate per l'espressione di diversi marcatori (CD90, CD45, CD44, CD34, CD31, Flk1, Sca1, CD105) attraverso la citometria a flusso e per l'espressione di Oct4, Sox2, c-Myc, Klf4, Nanog e Sca-1, attraverso qRT-PCR , a diversi stadi embrionali. Sono stati valutati il potenziale ematopoietico in vitro e la capacità di formare teratomi in topi Rag2-/-γc-/-. Per la riprogrammazione a cellule embrionali germinali, le cellule AFS sono state seminate su feeder layer di STO o Sl4-m220, mitoticamente inattivato, con il terreno per le cellule primordiali germinali, supplementato con LIF e il fattore per la crescita dei fibroblasti (bFGF) e successivamente con il terreno N2B27 2iLIF. Per il trattamento dei topi HSA-Cre, SmnF7/F7 le cellule AFS isolate da topi GFP+, sia isolate a fresco che coltivate, sono state iniettate nelle vena della coda e i topi sono stati sacrificati un mese dopo trapianto ed i muscoli analizzati mediante ematossilina ed eosina, tricromica di Masson e con immunofluorescenza per l'espressione di distrofina/GFP. Per studiare l'origine embrionale delle cellule AFS sono stati utilizzati due modelli murini: Oct4-GFP e TNAP-Cre. Per l'induzione alla pluripotenza, cellule ottenute dai feti Oct4-GFP son state trasfettate con i plasmidi del trasposone PB-TET contenente quattro geni (Oct4, Sox2, c-Myc e Klf4) sotto il controllo trascrizionale del promotore inducibile tetO2 tetraciclina/doxiciclina. L'espressione dei geni della pluripotenta è stata indotta con la doxiciclina. Le cellule iPS così ottenute sono state testate per l'espressione dei marcatori Nanog, SSEA-1 e per la positività alla fosfatasi alcalina. Risultati: Le cellule AFS c-kit+ murine variano in numero durante il corso della gestazione. Queste cellule esprimono i marcatori ematopoietici (CD45, CD34, Sca1), mesenchimali (CD90, CD105) insieme a Flk1, CD31, CD44. Sulla base dell'epressione di c-kit sono state identificate due popolazione cellulari: c-kithigh e c-kitlow, che mostrano anche una differente espressione dei marcatori sopracitati. Le cellule c-kitlow sono presenti in numero maggiore e durante il corso della gestazione diminuiscono mentre le c-kithigh aumentano. Entrambe le popolazioni hanno un potenziale ematopoieitco in vitro. Le cellule murine AFS esprimono a bassi livelli i geni Oct4 e Sox2 e ad alti livelli c-Myc e Klf4, ma la loro espressione cambia durante il corso della gestazione. La stessa analisi eseguita a livello di singola cellula ha mostrato che allo stadio E13.5 il 5% delle cellule co-esprime Oct4, Sox2 e Klf4. Le cellule AFS murine non formano teratoma. Negli esperimenti di terapia cellulare topi HSA-Cre, SmnF7/F7 non trattati, morivano all'età di 10 mesi mentre topi trattati con le cellule AFS o cellule da midollo osseo non frazionato avevano una sopravvivvenza del 75% e del 50%, rispettivamente. I topi trattati con le cellule AFS, mostravano un recuperano della forza muscolare rispetto agli animali non trattati (+75%). I muscoli degli animali trattati con le cellule AFS mostravano una morfologia normale con un numero basso di fibre rigeneranti (<1%) e una normale espressione delle distrofina che per il 37.86% (± 9.48%) era GFP+. A 15 mesi dal trattamento, gli animali trattati con cellule del midollo osseo avevano un maggior numero di fibre centro nucleate e una consistente infiltrazione di tessuto interstiziale e nessuna fibra GFP+. Gli animali trattati con le cellule AFS mostravano un fenotipo migliore e il 58.00% (± 2.43%) delle fibre muscolari era GFP+. Risultati simili sono stati ottenuti trattando topi HSA-Cre, SmnF7/F7 con cellule AFS espanse in vitro. Per cercare di differenziare le cellule AFS in cellule embrionali germinali pluripotenti abbiamo testato due protocolli ma nessuna colonia che ricordasse cellule embrionali germinali si è formata in vitro. Le cellule AFS isolate dai feti Oct4-GFP erano GFP negative. Il modello TNAP-Cre è risultato essere aspecifico, perchè TNAP non risultava essere espresso solo nelle cellule germinali primordiali. Negli esperimenti di riprogrammazione, alcuni cloni di iPS hanno mostrato di essere doxiciclina indipendenti, esprimevano il gene Oct4 endogeno ed erano positivi per i marcatori Nanog e SSEA1 e per la fosfatasi alcalina. Discussione: Le cellule murine AFS sono una popolazione eterogenea, le cui caratteristiche variano durante il corso della gestazione ed esprimono marcatori ematopoietici, mesenchimali e anche marcatori tipici delle cellule endoteliali. Il potenziale differenziativo delle due popolazioni c-kithigh e c-kitlow dovrà essere testato in vivo. L'analisi dell'espressione genica a livello di popolazione e a livello di singola cellula per i marcatori della pluripotenza ha confermato l'eterogeneità delle cellule AFS. Il trattamento degli animali HSA-Cre, SmnF7/F7 ha mostrato un potenziale miogenico delle cellule murine AFS, anche a lungo termine, suggerendo un interessante potenziale terapeutico. Le cellule AFS potrebbero contribuire a generare nuove fibre muscolari fondendosi con fibre esistenti o integrarsi nelle nicchia delle cellule staminali muscolari, ma maggiori esperimenti saranno necessari per valutare la validità dell'ipotesi. Anche le cellue AFS coltivate hanno dimostrato il mantenimento delle proprietà rigenerative. Questo studio suggerisce che le cellule AFS murine probabilmente non derivano da cellule primordiali germinali. Tuttavia è importante ricordare che il modello murino Oct4-GFP non è un modello di lineage tracking. Maggiori esperimenti saranno necessari per confermare questi risultati e per identificare l'origine delle cellule AFS. Le cellule iPS sono un promettente strumento di ricerca come modello di malattia o nella speranza di ottenere una sorgente di cellule per terapia. Qui è stato dimostrato come il sistema del PB può essere un valido metodo per la riprogrammazione delle cellule murine AFS. Questi sono solo risultati preliminari e maggiori esperimenti saranno necessari per completarne la caratterizzazione.

Amniotic Fluid Stem Cells (AFSCs) can be isolated from the amniotic fluid after amniocentesis that pregnant women undergo during the second trimester of pregnancy, this population has already been characterised and share common features between embryonic stem cells and adult mesenchymal stem cells. AFSCs are identified by specific markers; this study – as already published – focuses on the CD117 (c-Kit) positive fraction of AFSCs. Those cells are of great interest for regenerative medicine purposes considering their potential of differentiation and the relative constant availability, moreover retrieving prenatal autologous cells can offer new strategies for new-borns with congenital malformations or diseases. This study was intended to explore the possibility of isolating AFSCs at term of pregnancy (so at the third trimester, retrieving the amniotic fluid during delivery). Comparison on phenotype and test of potential for different lineages differentiation has been investigated on cells from both trimesters. These cells have a great potential also because they can be kept in culture and their number significantly increased for successive applications. Hypoxia is also a well-known factor that influence culture of stem cells, cultivating cells at lower oxygen tension has already demonstrated various beneficial effects on other stem cells so it has been decided to try this approach to ameliorate the expansion of AFSCs. In vivo experiments were performed to verify in vitro results on angiogenic potential of AFSCs.<br>Le cellule staminali del liquido amniotico (AFSCs) possono essere isolate dal liquido amniotico in seguito ad amniocentesi cui le donne incinte si sottopongono durante il secondo trimestre della gravidanza, questa popolazione è già stata caratterizzata e condivide proprietà comuni fra le cellule staminali embrionali e le cellule staminali adulte mesenchimali. Le AFSCs possono essere identificate tramite specifici marcatori; in questo studio – com’è già stato pubblicato – si è puntata l’attenzione sulla frazione positiva per CD117 (c-Kit). Queste cellule sono di grande interesse ai fini della medicina rigenerativa, considerando il loro potenziale di differenziamento e la relativa costante disponibilità, inoltre il recupero di cellule prenatali di origine autologa offre la possibilità di nuove strategie per curare i neonati con malformazioni congenite o altre malattie. In questo studio si è voluto esplorare la possibilità di isolare le AFSCs al termine della gravidanza (quindi al terzo trimestre, recuperando il liquido amniotico durante il parto). Sono stati investigati i fenotipi e le potenzialità differenziative di entrambi i trimestri. Queste cellule hanno anche un grande potenziale poiché possono essere mantenute in cultura e il loro numero significativamente aumentato per successive applicazioni. L’ipossia è un ben conosciuto fattore che influenza la coltura delle cellule staminali; coltivare le cellule in basse tensioni di ossigeno è già stato dimostrato avere un effetto benefico su altre cellule staminali, è stato quindi deciso di provare quest’approccio per migliorare l’espansione delle AFSCs. Sono stati effettuati esperimenti in vivo per verificare il potenziale angiogenico delle AFSCs.

A conventional 2D (two-dimensional) culture, in T-flasks or multi-well plates, is commonly perfo med for the stem cell development; however, it is time and labour consuming process. Moreover, it is impractical to scale-up to high cell number production. Growing stem cells inside bioreactor might be a solution. 3D bioreactor is not only a solution for scalable production but also a mimic environment for in vivo system. Herein, sparged-type bioreactors (e.g. airlift bioreactor) were chosen as bioreactors to differentiate murine embryonic stem cells (mESCs) into type II pneumocytes in the lung. There are two main sections in this thesis: the design of airlift bioreactor using computational fluid dynamics (CFD) and the differentiation of mESCs into the alveolar progenitor cells in a sparged bioreactor. The airlift bioreactors provide a better environment, which theoretically has been known to simulate the gas-exchange interface encountered in the lung alveoli. They require a low power input and provide a low shear environment with good mixing. The hydrodynamics (gas holdup, superficial liquid velocity, and shear rate) and mass transfer (kLa, the volumetric mass transfer coefficient) features of different airlift designs were determined by CFD. The simulations were based on a 3D transient model, Eulerian-Eulerian approach, and two-phase liquid/gas model with all phases being treated as laminar flow. The superficial gas velocity was varied from 0.001 m/s to 0.02 m/s. The simulation results indicated that the hydrodynamics were corresponded to the data found in literatures and the gas holdup were agreed with an experiment validation. The CFD results also suggested that in which range of superficial gas velocity (ug) that the system can be operated without any fluctuation in terms of the hydrodynamics. In addition, the airlift bioreactor is suitable for shear sensitive cells with high mass transfer rate, e.g. kLa, = 180 hr-1 at ug= 0.01 m/s and normoxia (20% O2) condition. Hence, the results from these simulations have been initially utilised as a promising hypothesis to design an airlift bioreactor for the scalable and automatable culture in multiphase bioreactors. For the second part, mESCs were encapsulated in a calcium-alginate hydrogel to create a 3D environment then the encapsulated cells weregrown in both 3D static culture, in a T-flasks, and the sparged bioreactor. The gas, 5% CO2 and 20% O2, was directly sparged into the bioreactor. The A549 conditioned medium was used to induced the mESCs to the endodermal lineages, targeting for the alveolar type II cells, type II pneumocytes. The differentiated cells expressed lung cell markers: SPC (pneumocyte type II), and FoxA2 (endoderm marker). In experiments, the relative expression of SPC markers reached the maximum level, 10-fold increase, at day 14 and day 20 for 3D static culture and the sparged bioreactor, respectively. After day 20 of the differentiation process, the pneumocyte-like cells in static culture trend to lose their SPC expression whereas the cells in sparged bioreactor maintain relatively high SPC markers. At the end of a differentiation protocol, day 30, it was observed that both systems highly expressed the endodermal makers, FoxA2, i.e. approximately 2000-fold increase for static culture and 5000-fold increase for the sparged bioreactor. In conclusion, the direct gassing in the sparged bioreactor not only enhanced the differentiation of embryonic stem cells into type II pneumocytes but also mimicked the in vivo environment in the lung therefore the differentiated cells can maintain the lung phenotype for a long term culture, up to 5 weeks in vitro culture. This in vitro system would be beneficial for drug screening and regenerative medicine applications.

Le cellule staminali mesenchimali derivate da liquido amniotico (AFMSC) sono ottime candidate per la terapia rigenerativa di patologie neurodegenerative grazie alle loro caratteristiche di facile isolabilità, staminalità e potenzialità immunomodulatrice. In questo studio sono state analizzate e indotte a differenziamento in senso neuronale AFMSC isolate da 9 campioni di liquido amniotico raccolto nel secondo trimestre di gravidanza. Le AFMSC sono state caratterizzate immunofenotipicamente e genotipicamente ed è stato valutato il loro potenziale differenziativo in senso osteogenico, condrogenico e adipogenico per definirne il carattere di staminalità. Le cellule isolate hanno soddisfatto tutti i criteri, dimostrando di essere cellule realmente staminali. Successivamente, è stato indotto il loro differenziamento in senso neuronale tramite co-coltura indiretta con cellule astrocitarie umane. Durante la co-coltura, le AFMSC hanno avuto un progressivo rallentamento del tasso di proliferazione cellulare e comparsa di morfologia neurite-like, significativa di una risposta ai fattori secreti dalle cellule gliali della co-coltura. L’analisi con RT-PCR (PCR Array) di AFMSC dopo co-coltura ha dimostrato notevoli variazioni di espressione dei geni regolatori del ciclo cellulare, della divisione simmetrica e asimmetrica, nei markers di autorinnovamento e di differenziazione cellulare, nelle citochine e nei fattori di crescita. Anche l’espressione dei geni coinvolti nella neurogenesi ha dimostrato notevoli variazioni dopo co-coltura con cellule della glia. I geni correlati all’autorinnovamento, invece, sono risultati inibiti. Le AFMSC dopo co-coltura presentano infine un aumento dell’espressione proteica di Nestina e β-Tubulina III, markers dei progenitori neurali, mentre non è stata riscontrata l’espressione dei markers di espressione gliale GFAP e S100. È possibile concludere che le AFMSCs sembrano rispondere al differenziamento neuronale, anche se ulteriori analisi di tipo elettrofisiologico saranno necessarie, ponendosi quindi come ottimi candidati per la terapia di patologie neurodegenerative.<br>Amniotic fluid mesenchymal stem cells (AFMSC) are great candidates for regenerative therapy of neurodegenerative diseases due to their extensive capability of self-renewal, differentiation in specialized cells, lack of ethical restriction and immunogenic potential. In this study, we analyzed and induced to neuronal differentiation AFMSC isolated from 9 samples of amniotic fluid collected in the second trimester of pregnancy. AFMSC have been characterized immunophenotypically and genotypically; to define AFMSC stemness characteristic osteogenic chondrogenic and adipogenic differentiative potential was assessed. The cells isolated from amniotic fluid matched all the stemness criteria, proving to be stem cells. Subsequently, their neuronal differentiation was induced with an indirect co-culture with human astrocyte cells. Co-cultured AFMSC had a gradual slowdown in cell proliferation rate and differentiate into neuron-like cells as a response to factors secreted by glial cells in co-culture. RT-PCR (PCR Array) analysis of co-cultured AFMSC showed significant changes in expression of cell cycle regulators genes, in symmetric and asymmetric division, in self-renewal and cell differentiation markers, in cytokines and growth factors. The expression of neurogenesis involved genes after co-culture with glial cells showed considerable variations too. The self renewal relates genes related were reduced. Co-cultured AFMSC showed an increased protein expression of Nestin and β-tubulin III, markers of neural progenitors and no expression of glial expression markers GFAP and S100. In conclusion, AFMSC seem to respond to neuronal differentiation, although further electrophysiological analysis will be necessary; therefore, AFMSC are excellent candidates for neurodegenerative diseases therapy.

Mesenchymal stromal cells (MSC) are multipotent cells found in fetal, neonatal and adult tissues. Fetal MSC have advantageous characteristics over their adult counterparts, and the regenerative potential of fetal blood MSC has recently been shown in a model of skeletal dysplasia and renal failure. Although fetal blood MSC can be isolated during ongoing pregnancy, the clinical effectiveness of using fetal blood-derived MSC for prenatal fetal cell therapy is constrained by the invasive nature of blood sampling procedure. With amniocentesis and chorionic villus sampling (CVS), fetal MSC can be obtained with minimal invasion. The aim of this study was to characterise stem cells from 1st trimester amniotic fluid (AF) and placenta by comparing their phenotype with MSC from 1st trimester bone marrow and 2nd trimester AF. Cells from all sources have similar immunophenotype, express pluripotency markers and telomerase, but 1st trimester AF stem cells have higher kinetics. The cells can differentiate into 3 lineages (bone, fat and cartilage), form embryoid bodies (EB) in vitro and can be transfected with high efficiency using non-viral methods. The migration potential of fetal MSC was also investigated using in vitro migration assays, to recapitulate the in vivo mechanisms involved in donor cell recruitment to various tissues and delineate the pathways involved. Fetal blood MSC and AF stem cells were shown to express CXCR4, the stromal cell-derived factor-1 (SDF-1) receptor, intracellularly but not on the cell membrane and migrate to SDF-1 gradients and to osteoblast cultures derived from the Osteogenesis Imperfecta mouse (oim), but not wild type bones. Pre-stimulation with oim plasma up-regulated CXCR4 and increased chemotaxis to SDF-1 and oim bone. Conclusively, 1st trimester AF and placenta are a new source of stem cells with great potential for future cell therapy applications. Also, initial experiments indicate the importance of the SDF-1/CXCR4 axis for stem cell recruitment to the site of injury.

Background. In the last years tissue engineering for cardiac pathologies has been broadly developed with the aim to restore or improve the diseased or damaged heart. Novel cardiac tissue engineering approaches combine the use of biocompatible scaffolds with stem cells to conjugate material science, surgery and cell therapy techniques. So far, different kinds of stem cells have been described and their potential for cardiac regeneration broadly investigated. We have previously described that it is possible to derive lines of broadly multipotent cells from the amniotic fluid (Amniotic Fluid Stem cells; AFS cells). The aim of this study was to characterize more in detail the AFS cells cardiomyogenic potential both in vitro and in vivo. Methods. Neonatal rat cardiomyocyte (rCM) cells were obtained by enzymatic digestion of 2-3-days old rat hearts. GFP-positive rat AFS (gfp+rAFS) cells were obtained from amniotic fluid samples from GFP-positive transgenic pregnant rats. Human AFS (hAFS) cells were obtained from healthy amniotic fluid back up samples from prenatal diagnosis, following informed consent. AFS cells were isolated by immunosorting for the stem marker c-kit. Before applying a tissue engineering approach, using biocompatible scaffolds, to the AFS and rCM cells coculture, the AFS cells “cardiomyocyte-like” phenotype, acquired in cocolture, had been functionally evaluated by patch-clamp analysis. In this work two different kinds of bidimensional micropatterned scaffolds were used: hydrogel films and PDMS (silicon) membranes. The scaffolds were obtained by microcontact printing technique and using a mold scratched with the desidered micropattern and their viability was tested using, at first, the rat neonatal primary culture. AFS and rCM cells were seeded together on the micropatterned PDMS membranes and analyzed for the expression of troponin T by immunostaining after 6 and 10 days of culture. For the in vivo study, immunodeficient nude male rats underwent a cryoinjury on the heart left ventricle with a 3D collagen scaffold implantation and 5x10e6 hAFS cells/animal local or systemic injection after 15 days. hAFS cells were previously labelled with the red intracellular fluorescent dye CMTMR. Animals were sacrificed at 24 hours, 15 and 30 days after cells injection and hearts stained for cardiac and inflammatory markers. For the acute myocardial infarct model, male Wistar rats underwent an ischemic injury by left anterior descendent coronary artery ligation for 30 minutes and then they were reperfused injecting via the external jugular vein 10e7 or 10e6 gfp+rAFS and 10e7 or 5x10e6 hAFS cells/animal for 2 hours; rats were sacrificed afterwards and hearts analyzed for infarct size measurement by Evans blue staining, by 2,3,5-triphenolltetrazolium chloride (TTC) staining and planimetry with the software Image J. Heart, lungs, spleen and liver were analyzed as well by immunostaining for evaluating hAFS cells content. hAFS cells were also analyzed for the presence of a subpopulation of cardiac progenitors, by RT-PCR analysis, for the expression of early cardiac commitment genes as Isl1 and Kdr. The cells were then studied by ELISA essay to speculate if they can secrete in the culture medium the protein thymosin beta 4, paracrine and cardioprotector factor. Results and Conclusions. Regarding the in vitro results, AFS cells were demonstrated to express a “pace maker cell-like” action potential, when cocultured with rat neonatal cardiomyocyte cells. Moreover, when cultured on the bidimensional scaffold, AFS cells showed to follow the longitudinal orientation of the microstruttured membrane, expressing beating activity and the cardiac protein troponin T. Our in vivo data revealed that hAFS cells, injected into the cryoinjured rat heart, survived in the host up to 30 days, moved from the injection site to the lesioned area in the heart and gave rise to new chimeric capillaries in the patch and cryoinjury area. In the acute myocardial infarct model the results obtained suggested that hAFS cells could exert a paracrine effect in vivo, decreasing the infarct size (measured as the ratio between the infarct area and the ischemic area at risk of necrosis) from a 53,9 ± 2,3% (obtained in control animals receiving PBS injection) to 40,0 ± 3,0% of the ischemic area. Furthermore, hAFS cells were also demonstrated to have a subpopulation of cardiac progenitors, positive for the expression of the early cardiac commitment genes Isl1 and Kdr and to to secrete in the culture medium thymosin beta 4, a paracrine factor previously shown to act as cardioprotector and angiogenic agent. In conclusions, our results are very encouraging and challenging, suggesting that AFS cells can show cardiomyogenic potential and cardioprotective therapeutic application in cell based therapy tissue engineering.

Thesis (M. S.)--Mechanical Engineering, Georgia Institute of Technology, 2010.<br>Committee Chair: Guldberg, Robert; Committee Member: McDevitt, Todd; Committee Member: Zamir, Evan. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Chronic kidney Disease (CKD) is a global health problem. It is associated with gradual decline in renal function, which develops into end stage renal disease and culminates into renal failure. Many different etiological factors are involved in the initiation and progression of CKD. The two most prevailing medical reasons for the development of progressive kidney disease are diabetes and hypertension. Regardless of the site of the initial insult, which may either be the glomerulus or tubules, all forms of progressive kidney diseases follow a final common pathway that is interstitial and glomerular fibrosis together with loss of podocytes, therefore, loss of glomerular integrity and renal function. Current therapeutic options for CKD are few and limited to the administration of renin-angiotensin system blockers, dialysis and renal transplantation. Although, pharmacotherapies are effective in slowing down the rate of progression, they do not prevent end stage renal disease. On the other hand dialysis even though life sustaining does not solve the problem and is associated with very poor quality of life. Renal transplantation thus remains the most effective method to treat end stage renal disease, however, the shortage of donor organs and immune complications associated with it do not solve the problem either. Hence, development of novel therapies remains an urgent necessity. Stem cell approach to Regenerative Medicine introduces vast possibilities for new therapies in acute as well as chronic diseases where current treatment options are either limited or inadequate. Stem cells derived from amniotic fluid (AFSC), which have emerged in recent years, represent a new source of stem cells with pluripotential properties, devoid of ethical issue associated with embryonic stem cells. Previous studies have proven the principle that AFSC can be used in regenerative medicine of the kidney due to their strong immunomodulatory properties and showed renoprotection when injected into an acute renal failure injury model in rodents. The purpose of the current study was to investigate the role of stem cells derived from amniotic fluid in an animal model of chronic kidney disease. In particular, cells positively selected for CD117 (c-kit) were administered to mice with x-linked Alport Syndrome (XLAS), a well established genetic model to study chronic kidney disease. Alport disease is associated with mutations in the collagen type IV family of proteins, the a3, a4 and a5 chains, the absence of which alters the normal composition and function of the glomerular basement membrane leading to proteinuria, and ultimately loss of podocytes. We hypothesized that (AFSC) would be able slow down chronic progression in Alport kidneys by modulating key processes such as the immune response, pro-fibrotic events initiated by TGF- signaling and cytokine/chemokine profile of the kidney via endocrine/paracrine mechanisms protecting the glomerular structure and preserving the filtration property of the organ. Single dose of systemic injection of AFSC at an early stage of the disease, before the onset of proteinuria, prolonged the life-span of Alport mice by 20% on average. This also resulted into amelioration of the functional parameters of the kidney, lowered serum creatinine, BUN as well as proteinuria levels at 2.5 months post treatment. Treated kidneys demonstrated better morphology with less severe glomerulosclerosis, lesser deposition of collagen type I associated with interstitial fibrosis and reduced infiltration of macrophages and inflammatory cells compared to their non-treated siblings. AFSC were detectable in glomeruli of injected mice but they did not differentiate into podocytes, and consequently, the a5(IV) chain of collagen missing in Alport mice was not replaced by AFSC treatment. Our findings revealed significant downregulation of key regulatory cytokines, such as TNFa, CCL2, CXCL2 and M-CSF involved in pro-fibrotic M1 macrophage signaling pathway favoring tissue remodeling instead of tissue injury. Finally, Alport mice receiving AFSC demonstrated preservation of podocyte numbers. In a closer look, glomerular cells stimulated with Angiotensin II showed increased Angiotensin II receptor type 1 (ANGTR1) expression, which was downregulated when treated with losartan (Angiotensin II antagonist). Similar to losartan AFSC decreased ANGTR1 expression, suggesting that AFSC may block the effects of Angiotensin II favoring glomerular survival. In conclusion, AFSC slow down Alport progression via preservation of podocyte number, M2c activation of macrophages and modulation of kidney microenvironment through endocrine/paracrine mechanisms favoring tissue preservation and function, in particular preserving podocyte number and filtration property.<br>Negli ultimi anni l’aumento nell’incidenza della Malattia Renale Cronica (Chronic Kidney Disease, CKD) è diventata un importante problema di salute pubblica. Principale manifestazione clinica della CKD è un graduale declino della capacità di filtrazione del rene che termina con lo sviluppo di insufficienza renale terminale (End Stage Renal Disease, ESRD). Infezioni, infiammazioni acute e croniche, malattie genetiche o la presenza di patologie a carico di altri organi, come per esempio il diabete, possono concorrere o essere causa diretta di danno renale. Questi insulti possono portare ad una progressiva perdita della funzione renale fino a raggiungere lo stadio finale di ESRD. Indipendentemente dalle origini eziologiche, la CKD evolve con la progressione di fibrosi glomerulare e interstiziale, apoptosi dei podociti con conseguente distruzione della struttura morfo-funzionale del rene e della sua capacità di filtrazione. L’utilizzo di farmaci (come per esempio gli antiipertensivi ACE inibitori, o inibitori dell'anidrasi carbonica) spesso riesce a rallentare il progresso della malattia ma il trattamento farmacologico risulta essere meno efficace con il progredire dell’ESRD e può risultate nefrotossico con il passare del tempo. La dialisi, trattamento molto invasivo e costoso per la sanità, ed il trapianto con la scarsa disponibilità di organi e la crescente richiesta, hanno spinto alla ricerca di valide alternative con un minor costo sociale ed una maggior compliance da parte dei pazienti. Molti studi si sono focalizzati nella scoperta di nuove fonti di cellule staminali per il trattamento di malattie renali acute e croniche dove le terapie correnti sono insufficienti ed inadeguate. Negli ultimi anni, il liquido amniotico è stato proposto come fonte alternativa per l’isolamento di cellule staminali da poter utilizzare in futuro per la terapia cellulare e, con particolare riguardo, per la rigenerazione del tessuto renale. In particolare, in studi recentemente pubblicati, le cellule staminali da liquido amniotico (AFSC) si sono distinte per la loro potente azione antiinfiammatoria e renoprotettiva, con modulazione della risposta immunitaria in un modello murino di danno renale acuto. Lo scopo di questo lavoro è quello di investigare il ruolo delle cellule staminali da liquido amniotico in un modello animale genetico di danno renale cronico (CKD). Nello specifico, cellule positive per il recettore di membrana CD117 (c-kit) sono state infuse in un modello murino di Sindrome di Alport, riconosciuto come uno dei più utili modelli più utili per lo studio di malattie renali croniche. La Sindrome di Alport è una grave malattia ereditaria e progressiva dovuta a mutazioni che coinvolgono le catene a3, a4 and a5 del collagene di tipo IV. L’assenza di tali catene altera la composizione fisiologica della matrice extracellulare renale (GBM, glomerular basement membrane) e provoca una marcata perdita di funzionalità che conduce a proteinuria e, con il tempo, a perdita dei podociti con conseguente blocco della filtrazione glomerulare. Basandoci sui dati precedentemente ottenuti, in questo lavoro ipotizziamo che le cellule staminali da liquido amniotico abbiano la capacità di rallentare la progressione della CKD, tramite un’azione endocrina e paracrina su vari meccanismi patologici che includono la risposta immunitaria, la fibrosi indotta dall’attivazione di TGF-a . In aggiunta, ipotizziamo che l’azione positiva esercitata dalle cellule da liquido amniotico sia un effetto di mantenimento della struttura glomerulare con conseguente mantenimento della normale capacità di filtrazione. In particolare, una singola infusione di AFSC, amministrata nei primi stadi della malattia, è capace di prolungare la sopravvivenza nel modello murino di circa il 20% con un significativo miglioramento dei parametri fisiologici renali quali livelli di creatinina plasmatica, BUN e proteinuria, oltre due mesi dalla somministrazione. Inoltre, Il tessuto renale degli animali trattati presenta una morfologia normale con lieve sclerosi glomerulare ed una minore deposizione di collagene I, di solito associata a fibrosi interstiziale. A questo si accompagna una minore infiltrazione da parte di macrofagi e altre cellule del sistema immunitario rispetto agli animali non trattati. Una vasta serie di esperimenti è stata quindi effettuata per investigare i meccanismi di azione tramite cui le cellule staminali da liquido amniotico esercitano il loro effetto positivo. Gli animali trattati presentano un maggior numero di podociti se comparati con i topi di controllo. La presenza di cellule infuse nei glomeruli nel tempo, non è stata però accompagnata da differenziazione in podociti e produzione di nuovo collagene, escludendo quindi che il maggiore numero di podociti sia dovuto a proliferazione e differenziazione delle cellule amniotiche. Dati ottenuti tramite esperimenti in vitro confermano l’effetto positivo delle AFSC nella risposta delle cellule del glomerulo all’Angiotensina II , tramite diminuzione nell’espressione del suo recettore di tipo 1 (ANGTR1), esercitando un effetto benefico di protezione delle cellule del golomerulo, in particolare dei podociti. Contemporaneamente, una significativa variazione nel profilo immunostimolatorio è stata rilevata negli animali trattati, che presentavano una diminuzione significativa nell’espressione di molecole quali TNF-a, CCL, CXCL2 e M-CSF, coinvolte nella stimolazione di macrofagi di fenotipo M1, noti per la loro azione pro-fibrotica e pro-infiammatoria. Per concludere, le cellule staminali da liquido amniotico sono capaci di rallentare la progressione della Sindrome di Alport tramite mantenimento del numero di podociti, attivazione di macrofagi M2c pro-rigenerativi a discapito dei pro-fibrotici M1 e modulazione endocrina/paracrina dei segnali cellulari all’interno del rene; favorendo il mantenimento strutturale e funzionale del tessuto renale.

Amniotic fluid stem cells (hAFSC) are emerging as a potential therapeutic approach for various disorders. The low number of available hAFSC requires their ex vivo expansion prior to clinical use, however, during their in vitro culture, hAFSC quickly reach replicative senescence. The principal aim of this study was to investigate the aging process occurring during in vitro expansion of hAFSC, focusing on the redox control that has been reported to be affected in premature and physiological aging. My results show that a strong heterogeneity is present among samples that reflects their different behaviour in culture. I identified three proteins, namely Nox4, prelamin A and PML, which expression increases during hAFSC aging process and could be used as new biomarkers to screen the samples. Furthermore, I found that Nox4 degradation is regulated by sumoylation via proteasome and involves interactions with PML bodies and prelamin A. Since various studies revealed that donor-dependent differences could be explained by cell-to-cell variation within each patient, I studied in deep this phenomenon. I showed that the heterogeneity among samples is also accompanied by a strong intra-population heterogeneity. Separation of hAFSC subpopulations from the same donor, using Celector® technology, showed that an enrichment in the last eluted fraction could improve hAFSC application in regenerative medicine. One of the other problems is that nowadays hAFSC are expanded under atmospheric O2 concentration, which is higher than the O2 tension in their natural niches. This higher O2 concentration might cause environmental stress to the in vitro cultured hAFSCs and accelerate their aging process. Here, I showed that prolonged low oxygen tension exposure preserves different hAFSC stemness properties. In conclusion, my study pointed different approaches to improve in vitro hAFSC expansion and manipulation with the purpose to land at stem cell therapy.

Mesenchymal stem cells have been recently investigated for their potential use in regenerative medicine. It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Moreover, population of adult stem cells were recently identified in human and lab animal tendons, but no detailed investigations have been made in the equine species. The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs), umbilical cord matrix (EUC-MSCs) and tendon derived progenitor cells (TSPCs). This work defines a protocol for extraction, isolation, expansion and characterization of mesenchymal stem cells from equine bone marrow, amniotic fluid, umbilical cord matrix (Wharton’s jelly) and tendon. Their localization into the tissues from which they were extracted, was reported. During the cell culture, cell expansion, CFU-F assay, doubling time, plasticity and immunophenotype were analyzed. Furthermore, a specific cell labeling was realized that could be used for non-invasive magnetic resonance cell tracking through endosomal incorporation of superparamagnetic iron oxide particles through the in vitro evaluation of the efficiency of this labeling method. In the next future, this technique should facilitate translation of the approach into clinical trials, in particular to track cells in vivo after transplantation and to follow their homing, viability and repair potential during time. The mesenchymal stem cells were grown on control medium, such as DMEM with the addition of basic FGF. Our results pointed out that these cells performed similarly in terms of CFU-F formation and growth kinetic. The immunocytochemical and RT-PCR analysis of MSCs isolated from all tissues showed the presence of antigens such as CD44, CD105, CD29, Oct-4, c-Myc, SSEA4 and HLA-ABC, whereas they were negative for CD34 and HLA-DR. These cells, differentiated into osteogenic, adipogenic, chondrogenic and tenogenic lineages confirming the nature of mesenchymal stem cells. These findings suggest that AF-MSCs appeared to be a readily obtainable and high proliferative cell line that may represent a good model system for stem cell biology. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation. Equine TSPCs have high clonogenic properties and proliferating potential, they express stem cell markers and have the capability to be multipotent as well as BM-MSCs. These findings suggest that TSPCs may represent a good model for stem cell biology and could be useful for future tendon regenerative medicine investigations. These data could be useful for optimization of horse’s mesenchymal stem cell isolation and expansion protocol that could be used in the experimental clinic application for tissue regeneration because to their biological properties. The MSCs isolated from equine extra-embryonic tissues and tendons, showed characteristics of stem cells and therefore can be regarded as good candidates to be used in regenerative medicine, with special reference to orthopaedic diseases of horses. Furthermore, future investigations in vivo are useful to evaluate the efficacy of cell labeling with contrast agents for magnetic resonance with particular attention to the muscle-skeletal tissues.

Spinal cord injury (SCI) is a disabling degenerative disease that heavily impacts on patient's life. To date, treatments available are mostly vain, thought to target the inflammatory processes sustaining the secondary damage: in this scenario, the use of stem cells represents a promising new therapeutic strategy as it might permit a complete recovery of the patient through a regeneration of damaged nerve fibers. Among the different type of stem cells available, we focused on 3° trimester amniotic fluid-derived cells (Afs), verifying their potential therapeutic activity on a mouse model of SCI. Only the cultures expressing NG2 were able to elicit an improvement in the motor performance, obtained through a better preservation of damaged tissue. It was observed a higher angiogenesis in the perilesion area, sustained by an upregulation of the pro-angiogenic genes HIF-1α and VEGF; furthermore macrophage infiltration levels were significantly reduced in cells treated mice compared to controls. The Hepatocyte Growth Factor (HGF) mRNA and protein levels resulted higher in the filtering organs, as the lungs, and in the bloodstream of transplanted animals, suggesting an endocrine effect of the cells. The ability of the cells to produce HGF was confirmed in vitro, after stimulation with proinflammatory cytokines, like IL1-β and LPS, suggesting the cells might be responsive to an environment-induced activation. Given these results, AFs demonstrated to exert a therapeutic effect in our animal model of spinal cord injury.

Type I diabetes (T1D), also known as diabetes mellitus or juvenile diabetes, is a metabolic disorder characterized by the selective destruction of pancreatic β cells of the islets of Langerhans. This loss, caused by an autoimmune attack of the endocrine cells, leads to hypoinsulinemia and hyperglycemia. T1D has nowadays reached epidemic proportions and the number of patients is rapidly increasing. To date, treatments are limited to administration of exogenous insulin and islets transplantation therapy, whose widespread applicability is though limited by scarcity of donors and risks related to immunosuppressive regimens. Stem cell therapy could represent a reliable treatment in the near future. In the context of regenerative medicine, stem cells have garnered much attention as a new potent source for the treatment of severe pathologies in which a specific tissue is lost or damaged, as in diabetes. Both embryonic and mesenchymal stem cells have been frequently investigated and represent an alternative source of in vitro-differentiated implantable β cells or can be directly used in stem cell therapy treatments. Different studies have already reported the application of mesenchymal stem cell (MSC) therapy for the treatment of autoimmune disease as T1D, showing that tissue protection and repair are obtained primarily via paracrine immunomodulatory functions. Amniotic fluid represents a new source of pluripotent stem cells. It contains a heterogeneous population of cells originating from the developing fetus, including c-kit positive cells, which exhibit both embryonic and mesenchymal stem cell characteristics. Amniotic fluid stem cells (AFSC) can be easily isolated without causing any harm to the fetus, thus there is no ethical concern in their use, can be extensively expanded in vitro and have the advantage of not forming tumors in vivo. Our laboratory has already demonstrated the successful therapeutic application of AFSC in a murine model of acute and chronic kidney injury as well as in model of acute and chronic lung injury. The aim of the project was to test the therapeutic potential of AFSC for the treatment of an acute mouse model of T1D. We hypothesize that intracardiac injections of AFSC on diabetic mice have the potential to restore pancreatic functional loss and to protect the pancreatic tissue from further damage. Our approach was based on the in vivo injection of human AFSC into immunodeficient NOD/SCID mice. Mice were rendered diabetic by selective drug treatment with streptozotocin. Injected mice were divided into two distinct groups, the first one receiving a single cell injection and the second group receiving a double dose of cells. This approach was chosen in order to evaluate whether the effect of stem cells could depend from the frequency of injections. Mice treated with AFSC were compared to not-treated diabetic mice and wild type mice. Mice were constantly monitored for blood glucose levels and in all experimental groups that received AFSC some of the mice maintained normoglycemic values during the time frame of investigation. Immunohistochemical assays reveal that diabetic mice treated with AFSC had significantly higher levels of insulin when compared to diabetic mice. Moreover, islets from AFSC-treated mice showed morphology similar to that of wild type islets. Integration of AFSC into the target organ was as well evaluated by immunofluorescence. However, despite the presence of some AFSC into the host tissue, the percentage of integrated cells resulted very low. Our hypothesis is that AFSC have the ability to restore pancreas functionality by mainly protecting endogenous beta cells, thus modifying the local microenvironment and/or by stimulating regeneration from endogenous progenitors rather than differentiate themselves into functional beta cells. In summary, we were able to show that AFSC can hold the potential to treat a severe condition such as diabetes by mainly protecting endogenous cells from damage. However the response to cell treatment is not equal among mice. We speculate that differences in outcomes among the mice can be due mainly to the type of injury that we performed, creating variations between different animals in the early phase of disease development.<br>Il diabete di tipo I, noto anche come diabete mellito o diabete giovanile, è una malattia metabolica caratterizzata dalla distruzione selettiva delle cellule β che risiedono nelle isole del Langherans del pancreas. Tale perdita, causata da un attacco autoimmune da parte di linfociti T e macrofagi che infiltrano il tessuto endocrino del pancreas, ha come immediata conseguenza lo sviluppo delle condizioni di ipoinsulinemia e relativa iperglicemia. Ad oggi, il diabete mellito ha raggiunto proporzioni epidemiche ed il numero di persone affette è in rapida espansione. Le attuali terapie per il trattamento del diabete mellito si limitano alla somministrazione d’ insulina esogena e, in alcuni casi, al trapianto di insule pancreatiche da donatore. Entrambe tuttavia presentano alcuni aspetti negativi tra cui la difficoltà nel mantenere sotto stretto controllo costante il livello di glucosio nel sangue, nel caso della terapia insulinica, ed il numero ridotto di donatori disponibili, nonché i rischi legati alle terapie immunosoppressive, per quanto riguarda il trapianto. Negli anni più recenti, la ricerca nel campo della medicina rigenerativa si è concentrata sulla possibilità di utilizzare le cellule staminali come fonte alternativa per la rigenerazione in vivo o in vitro di cellule che producano insulina e possano rimpiazzarne la perdita negli individui affetti. Sia le cellule staminali embrionali che mesenchimali sono state già analizzate e studiate come fonte alternativa di cellule da cui derivare in vitro beta cellule impiantabili o per un loro utilizzo diretto in trattamenti basati sulla terapia cellulare. Diversi studi hanno già riportato l'applicazione di terapie basate sull'uso di staminali mesenchimali per il trattamento di malattie autoimmuni quali il diabete mellito, dimostrando come la protezione e la riparazione del tessuto si ottengano principalmente mediante funzioni paracrine immunomodulatorie. Il liquido amniotico rappresenta una fonte nuova e alternativa di cellule staminali pluripotenti in quanto contenente una popolazione eterogenea di cellule che derivano dal feto in via di sviluppo, incluse le cellule positive per il fattore c-kit, le quali rappresentano circa lo 0.8-1 % dell'intera popolazione. Le cellule staminali da liquido amniotico (AFSC), positive per c-kit, possiedono caratteristiche delle cellule staminali embrionali e mesenchimali. L'uso delle AFSC presenta alcuni vantaggi tra cui la facilità di isolamento da campioni di liquido amniotico senza danno per il feto, il che permette di aggirare i problemi etici legati all'uso delle staminali embrionali. Inoltre, le AFSC possono essere espanse in vitro per numerosi passaggi ed è stato dimostrato, mediante la loro applicazione in vivo, che le AFSC non hanno proprietà tumorigeniche. Il potenziale terapeutico delle AFSC nel campo della medicina rigenerativa è già stato dimostrato con successo in modelli murini di danno, acuto e cronico, in rene e polmone. Lo scopo del progetto è la valutazione del potenziale terapeutico delle AFSC per il trattamento di un modello murino acuto di diabete mellito. L'ipotesi è che le AFSC, iniettate per via intracardiaca in topi diabetici, abbiano il potenziale di proteggere il tessuto pancreatico dal danno e favorire la rigenerazione. Il nostro approccio si è basato essenzialmente sull'applicazione in vivo di AFSC umane mediante iniezione intracardiaca in topi immunodeficienti NOD/SCID. Il diabete è stato indotto mediante trattamento chimico selettivo con streptozotocina ed i topi sono stati successivamente divisi in due gruppi al fine di valutare l'effetto di una singola iniezione di AFSC o di una doppia iniezione. I topi trattati con AFSC sono stati confrontati con topi diabetici e topi wild type. Gli animali sono stati monitorati ad intervalli di tempo regolari per quanto riguarda il parametro fisiologico del glucosio sanguigno e, al termine dell'esperimento, sacrificati. Solo alcuni dei topi trattati hanno risposto al trattamento con le staminali da liquido amniotico, mantenendo valori normoglicemici pressoché per tutta la durata dell'esperimento. Saggi immunoistochimici hanno permesso di evidenziare che i livelli di insulina di topi trattati con streptozotocina e iniettati con AFSC sono risultati significativamente superiori a quelli dei topi diabetici di controllo. Inoltre, le isole pancreatiche dei topi trattati con AFSC mostrano una morfologia molto simile a quella dei topi wild type. La percentuale di integrazione delle AFSC nel tessuto dell'ospite è risultata piuttosto scarsa, come già emerso in precedenti studi, supportando l'ipotesi secondo cui le AFSC abbiano la capacità di ripristinare la funzionalità pancreatica principalmente mediante protezione delle cellule beta endogene ed eventualmente stimolando la rigenerazione a partire da precursori endogeni piuttosto che per differenziamento diretto. Con l'approccio in vivo è stato possibile ottenere risultati preliminari promettenti per una futura applicazione di terapia cellulare possibilmente in ambito clinico. Tuttavia, il responso al trattamento non è uguale per tutti gli animali. Ipotizziamo che differenze nella risposta alla terapia cellulare possano dipendere dal tipo di danno acuto indotto, creando variazioni tra i topi nella prima fase di sviluppo della malattia.

Background. Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in neonates. No specific therapy is available and the treatment is only supportive. Amniotic Fluid Stem (AFS) cells represent a novel class of pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, as they are able to differentiate into lineages representative of all embryonic germ layers and do not form tumors after implantation in vivo. These characteristics, together with the absence of ethical issues concerning their obtainment, make AFS cells good candidates for cell therapy of human diseases. Aim. The aim of this study was to explore the therapeutic potential of Amniotic Fluid Stem (AFS) cells in a rat model of NEC. Methods. AFS cells were obtained from green fluorescent (GFP+) transgenic pregnant rats at 16 days p.c. by c-kit selection. NEC was induced in newborn rats by hyperosmolar milk formula, oral lipopolysaccharide and hypoxia. Rats were divided into 2 groups receiving at 24 and 48 hours of life an intraperitoneal injection of: (i) phosphate buffered saline (PBS; n=120) or (ii) 2x106 AFS cells (n= 121). Additional groups of animals, either injected with bone marrow-derived mesenchymal stem cells (i.e. rat BM-MSCs) or committed cells (i.e. rat myoblasts), or non subjected to NEC induction (i.e. healthy breast fed newborn rats), were used as additional controls. All groups were blindly compared regarding survival, clinical status, radiological features (abdominal MRI), gut motility (carmine red transit time) and intestinal permeability (plasma lactulose/mannitol ratio). Intestines were blindly analyzed for macro- and microscopic appearance, transcriptional profile (microarray-based expression analysis), neutrophil infiltration (myeloperoxidase activity), enterocyte proliferation (EdU assay) and apoptosis (cleaved caspase 3 immunohistochemistry). AFS cell integration in the gut was evaluated by GFP amplification and immunostaining. Cyclooxygenase 2 (COX2+) cells in the lamina propria were evaluated by immunofluorescence. COX2 activity was inhibited in vivo using selective (celecoxib) and non-selective (ibuprofen) inhibitors; the effects of COX2 pharmacological inhibition on rat survival and clinical status were evaluated. Results. Compared to animals injected with PBS, rats receiving AFS cells survived longer (p<0.0001), and showed: improved clinical conditions (p<0.001), better abdominal appearance at MRI, restored intestinal transit (p<0.01), decreased intestinal permeability (p<0.05), reduced macroscopical (p<0.001) and histological gut damage (p<0.001). These beneficial effects were specific to AFS cells as neither BM-MSCs nor myoblasts were able to improve animal morbidity/mortality in comparison to PBS (p=n.s.). AFS cells integrated in the intestine with various degrees of spreading in all the animals. cDNA arrays comparing the intestinal transcriptional profile of PBS vs. AFS cell rats showed differences in the expression of genes involved in inflammation, apoptosis and cell proliferation which were respectively down-regulated (inflammation and apoptosis) and up-regulated (proliferation) in the AFS cell group. At a protein level, AFS cell rats had lower gut neutrophil infiltration (p<0.05), reduced enterocyte apoptosis (p<0.05) and increased enterocyte proliferation (p<0.0001) compared to PBS rats. In rats treated with AFS cells vs. rats injected with PBS, COX2+ cells in the lamina propria were increased (p<0.001) and repositioned under crypts (p<0.001). Moreover, both the total number of COX-2+ cells per villus unit and the number of cryptal COX-2+ cells inversely correlated with the degree of intestinal damage (p=0.014). The pharmacological inhibition of COX2 activity did not exert any effect in PBS rats, whereas it completely abolished AFS cell beneficial effects on animal survival and clinical behavior. Conclusions. In experimental NEC, AFS cell administration via the intraperitoneal route is associated with reduced animal morbidity/mortality and decreased incidence of NEC. AFS cell beneficial effects seems to be related to decreased intestinal neutrophil infiltration, enhanced enterocyte proliferation and reduced epithelial apoptosis. We hypothesize that is achieved through activation of COX2+ cells in the lamina propria. Stem cell therapy may represent a new therapeutic option for infants with NEC.<br>Premesse. L’enterocolite necrotizzante (NEC) rappresenta la causa più frequente di insufficienza intestinale in età pediatrica. Non esistono tuttora terapie specifiche per la NEC ed il suo trattamento si basa unicamente sulla terapia medica di supporto e sulla rimozione chirurgica delle porzioni di intestino affetto. Le cellule staminali derivanti da liquido amniotico (AFSC) sono una popolazione di cellule staminali di origine fetale descritta per la prima volta nel 2007. Esse possiedono delle caratteristiche intermedie fra le cellule staminali embrionali (i.e. pluripotenza) e le cellule staminali adulte (i.e. mancata tumorigenicità dopo iniezione in vivo) che le rendono candidati ideali per la terapia cellulare. Scopo dello studio. Valutare il potenziale terapeutico delle cellule staminali derivanti da liquido amniotico (AFSC) in un modello animale di NEC. Materiali e metodi. AFSC sono state derivate da ratti Sprague-Dawley GFP+ (i.e. esprimenti in modo costitutivo la proteina reporter “Green Fluorescent Protein”) al 16^ giorno p.c. tramite immunoselezione per il loro caratterstico marcatore di superficie (i.e. c-kit/CD117). Le cellule ottenute sono state caratterizzate per morfologia e immunofenotipo. La NEC e’ stata indotta in ratti neonati tramite l’utilizzo di elementi simili ai fattori patogenetici implicati nell’insorgenza della NEC umana: alimentazione con latte formulato iperosmolare, eventi ipossici, somministrazione di lipopolisaccaride. I ratti, suddivisi in due gruppi principali, hanno ricevuto a 24 e 48 ore di vita, per via intraperitoneale: i. 50 ul di soluzione salina (PBS; n=120) o ii. 2x106 AFSC (n= 121). Altri gruppi di animali, trattati con cellule staminali mesenchimali di ratto derivanti da midollo osseo o con mioblasti, oppure non sottoposti all’induzione di NEC (i.e. neonati sani allattati al seno), sono stati utilizzati come gruppi aggiuntivi di controllo. I diversi gruppi di animali sono stati valutati in cieco per i seguenti parametri: sopravvivenza, stato clinico, aspetto radiologico intestinale (RM ad alta risoluzione), motilita’ intestinale (studio del tempo di transito con coloranti vitali), permeabilita’ intestinale (rapporto lattulosio/mannitolo plasmatici). L’intestino e’ stato valutato in cieco per: aspetto macroscopico ed istologico, profilo di espressione genica (tramite tecnologia cDNA-microarray), infiltrazione neutrofila (saggio di attivita’ della mieloperossidasi), proliferazione (EdU) e apoptosi degli enterociti (immunoistochimica per caspasi 3 attivata). L’integrazione di AFSC nell’intestino e’ stata analizzata sia tramite PCR (amplificazione del gene gfp) che tramite immunoistochimica (immunofluorescenza per GFP). Il numero e la localizzazione delle cellule stromali esprimenti COX2 nella mucosa sono stati valutati con immunofluorescenza. L’attivita’ di COX2, in vivo, e’ stata inibita farmacologicamente con inibitori selettivi (celecoxib) e non selettivi di COX2 (ibuprofene); gli effetti di tale inibizione sulla sopravvivenza e sulla morbidita’ degli animali trattai con AFSC o PBS sono stati analizzati in cieco. Risultati. La somministrazione di AFSC, per via intraperitoneale a ratti neonati affetti da NEC: migliora significativamente la sopravvivenza degli animali sia rispetto alla somministrazione di PBS (p<0.0001) che di linee cellulari di controllo (i.e. cellule staminali mesenchimali di ratto derivanti da midollo osseo [p=0.024] e mioblasti di ratto [p<0.0001]). Rispetto alla somministrazione di PBS, inoltre, il trattamento con AFSC: i. riduce la morbidita’ degli animali migliorandone l’aspetto clinico (p<0.001); ii. riduce significativamente il danno intestinale sia alla valutazione dell’addome con RM ad alta risoluzione che all’esame macroscopico (p<0.001) ed istologico dell’intestino (p<0.001); iii. migliora significativamente la funzionalità dell’intestino sia per quanto concerne la motilità (p<0.01) che l’assorbimento di nutrienti (p<0.05). AFSC somministrate per via intraperitoneale migrano preferenzialmente verso l’intestino dove, seppur con un basso tasso di integrazione tissutale, sono in grado di localizzarsi in tutti gli strati della parete e talora di differenziarsi in cellule con fenotipo mesenchimale (i.e. cellule muscolari lisce). La somministrazione di AFSC in ratti neonati affetti da NEC è in grado di modificare il profilo di espressione genica dell’intestino incrementando l’espressione di geni coinvolti nella proliferazione e riducendo l’espressione di geni coinvolti in apoptosi e infiammazione. Tali dati di espressione sono stati confermati a livello proteico dimostrando che nell’intestino dei ratti affetti da NEC trattati con AFSC v.s. PBS è maggiore la proliferazione delle cellule epiteliali (p<0.0001), minore l’apoptosi degli enterociti (p<0.05) e ridotta l’infiltrazione neutrofila tissutale (p<0.05). La somministrazione di AFSC, inoltre, determina l’attivazione di una popolazione di cellule stromali esprimenti la ciclossigenasi 2 (COX2) nella lamina propria della mucosa intestinale. Più in dettaglio la somministrazione di AFSC v.s. PBS causa un significativo aumento del numero delle cellule COX2+ nella lamina propria (p<0.001) e un loro spostamento dall’asse del villo alla niche delle cripte intestinali (p<0.001). Tale effetto costituisce il meccanismo d’azione di AFSC poiché la somministrazione in vivo di inibitori selettivi e non selettivi di COX2 (ma non di COX1) a ratti affetti da NEC abolisce gli effetti positivi di AFSC su morbidità e mortalità degli animali ma non ha alcun effetto sugli animali trattati con PBS. Conclusioni. In un modello animale di NEC, AFSC sono in grado di migliorare in modo significativo la mortalita’ e la morbidita’ degli animali e il danno intestinale. AFSC non determinano direttamente tali effetti rigenerando di per sé l’intestino ma indirettamente attivando le cellule stromali esprimenti COX2 presenti nella lamina propria le quali a loro volta stimolano la proliferazione e riducono l’apoptosi delle cellule epiteliali intestinali residenti. Sebbene ulteriori studi siano necessari (e.g. per identificare i fattori/meccanismi molecolari responsabili dell’attivazione delle cellule COX2+), riteniamo che la terapia con cellule staminali derivanti da liquido amniotico possa rappresentare una nuova prospettiva terapeutica per i pazienti affetti da NEC.

O cão é um excelente modelo pré-clínico para o estudo de doenças, testes farmacológicos e novas terapias para futura aplicação em humanos. Desta forma, estudamos o modelo canino como fonte de células tronco de anexos fetais, o líquido amniótico, alantóide e conteúdo vitelino. Uma vez que hoje, as células tronco apresentam uma esperança na cura de diversas doenças tanto nos cães, como no ser humano. Sendo assim, caracterizamos e estudamos o potencial de diferenciação dessas células, isoladas após a técnica de ovário salpingo histerectomia, de cadelas em campanhas de castração. Após o isolamento e a caracterização, somente foi estabelecida a cultura do líquido amniótico e alantóide. Para caracterizar as células, isoladas no intuito de comprovar que são células tronco verdadeiras, os seguintes Imunomarcadores foram usados, vimentina, nestina, citoqueratina-18 e oct-4, sendo os três primeiros positivos para células do líquido amniótico e alantóide. Induzimos a diferenciação dessas células para osso, cartilagem e gordura, utilizando protocolos previamente estabelecidos. As células tronco do líquido amniótico limitaram-se à diferenciação condrogênica e osteogênica enquanto que as células tronco do alantóide, limitaram-se a diferenciação condrogênica. Ao mesmo tempo, ambos os tipos celulares, não prosseguiram à diferenciação adipogênica. Surpreendentemente, o meio de diferenciação para gordura, induziu a mudança morfológica nestas células que passaram a apresentar a morfologia típica de células neuronais. Podemos concluir que provavelmente para diferenciação adipogênica, é preciso desenvolver outro meio de cultura, por outro lado, esse resultado sugere que devemos explorar o potencial neurogênico desses tipos celulares.<br>The dog is an excellent preclinical model for the study of diseases, pharmacological tests and new therapies for future application in humans. Thus, we studied the canine model as a source of stem cells from fetal membranes, amniotic fluid, allantois and fluid yolk. Since today, stem cells have a hope in curing various diseases in both dogs and humans. Therefore, we characterize and study the differentiation potential of these cells, isolated after the technique of ovarian salpingo hysterectomy. After the isolation and characterization, was only established the culture of amniotic and allantois fluid. To characterize the cells isolated in order to demonstrate that they are true stem cells, the following were used antibodies, vimentin, nestin, cytokeratin-18 and oct-4. The cells were reactive positively to vimentin, nestin, cytokeratin. We induced the differentiation of these cells osteogenic, chondrogenic, adipogenic, using previously established protocols. Stem cells from amniotic fluid were restricted to chondrogenic and osteogenic differentiation while the stem cells of the allantois, limited to chondrogenic differentiation. At the same time, both cell types, were not able to adipogenic differentiation. Surprisingly, the means of adipogenic differentiation, induced the typical morphology of neuronal cells. We can conclude that probably for adipogenic differentiation, we must develop other culture media, on the other hand, this result suggests that we should explore the neurogenic potential these cell types.

La medicina “rigenerativa” ha come scopo quello di riparare o ripristinare la funzionalità di organi o tessuti danneggiati da malattie, traumi o dal “semplice” invecchiamento mediante l’utilizzo di cellule staminali. Una linea cellulare staminale molto importante e molto utilizzata in approcci di medicina rigenerativa è costituita dalle staminali adulte o staminali mesenchimali. Il midollo osseo e il tessuto adiposo sono stati considerati, per lungo tempo, la principale fonte di staminali mesenchimali adulte. Tuttavia le staminali adulte prevedono tecniche di prelievo invasive, non prive di complicazioni, e la coltura richiede diverse settimane; inoltre, possiedono limitate capacità di proliferazione e differenziaamento che sono inversamente proporzionali all’età del donatore. Da qui l’interesse, per il clinico, di nuove fonti cellulari. La recente scoperta in campo umano dell’esistenza di staminali mesenchimali nei tessuti fetali ha reso queste cellule molto interessanti agli occhi dell’intera comunità scientifica. I tessuti fetali sono di facile accesso perchè solitamente eliminati alla nascita, non richiedono procedure invasive, il loro utilizzo non suscita controversie etico-morali e inoltre le staminali isolte da tali tessuti sono caratterizzate da proliferazione più rapida, una maggiore espansione in vitro e da un notevole potenziale differenziativo. Alla luce di queste considerazioni, l’obiettivo principale di questo studio è stato quello di recuperare cellule staminali mesenchimali da tessuti fetali umani extraembrionali (il villo coriale, il liquido amniotico e la membrana amniotica) durante 3 diverse epoche gestazionali e mantenerle in coltura per un lungo periodo. Allo scopo di verificare se l’età gestazionale influenzasse le proprietà di staminalità, le staminali isolate da tali tessuti sono state confrontate nel fenotipo, nella espressione del markers specifici di pluripotenza e mesenchimalità, nel potenziale proliferativo, differenziativo e immunosoppressivo e immunomodulatorio.<br>Regenerative medicine involves the use of living cells to repair, replace or restore normal function to injured tissues and organ. With the field of mesenchymal stem cell (MSCs) research taking off in the late 1980’s and the early 1990’s, scientists highlighted the importance of adult MSCs for regenerative medicine applications, and identified their presence in all adult tissues. Among MSCs, MSCs derived from fetal tissues (bone marrow, blood and liver) and extra-embryonic compartments (amniotic fluid, umbilical cord blood, amniotic membrane, chorion and placenta) are promising in cell-based therapies because of their beneficial properties in wound healing. Moreover, these tissues are ideal sources for studying the features of stem cell characteristics due to the possibility of harvesting large amount of tissue, without posing ethical debate, following prenatal diagnosis (as in the case of chorion from chorionic villi sampling and amniotic fluid from amniocentesis) or at birth (as in the case of amniotic membrane). In addition, MSCs isolated from fetal sources non only fulfill general characteristic of MSCs but exhibit features of embryonic stem cells including the expression of specific pluripotent markers. In fact, they possess higher proliferation rates, lower immunogenicity and wider differentiation capacity than their adult counterparts as well as immuno- suppressive and modulatory properties. Scarce information on the behavior of MSCs from different stages of human gestation are so far available. The first aim of this study was the isolation of MSCs from fetal (extra-embryonic) tissues during first- second- and third- gestation period and their long-term culturing; secondly, the detection of the common features between chorionic villi (CV), amniotic fluid (AF) and amniotic membrane (AM)-derived MSCs; thirdly, the observation of differences in phenotype, proliferative capacity, differentiation ability as well as in the immuno-suppressive/immuno-modulatory properties among them.

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by time-varying flow environments, play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). There is strong evidence to support this hypothesis in tissue engineering studies of bone. From observing native heart valve dynamics, OSS can be created by means of pulsatility or by cyclic specimen geometry changes. However, quantification of the individual or combined effects of these variables for the maximization of OSS environments in vitro is to date, not known. Accordingly, in this study we examined and quantified the role that i) physiologically relevant scales of pulsatility and ii) changes in geometry as a function of specimen flexure, have in creating OSS conditions for dynamic culture of tissue. A u-shaped custom made bioreactor capable of producing flow stretch and flexure was used. Computational Fluid Dynamic (CFD) simulations were performed through Ansys CFX (Ansys, Pittsburgh, PA) for both steady and pulsatile flow. We have shown that OSS can be maximized by inducing pulsatile flow over straight scaffolds. We believe that OSS promotes BMSCs tissue formation.

Les cellules souches mésenchymateuses (CSM) interviennent de plus en plus dans le domaine de la médecine régénérative, notamment pour traiter des maladies aujourd’hui difficilement curables avec les moyens actuels. Deux verrous scientifiques limitent pourtant leur utilisation et leur commercialisation. D’une part, de grandes quantités de cellules sont nécessaires pour répondre à la forte demande médicale. D’autre part, les cellules étant elles-mêmes le médicament final, délivré chez le patient, leur qualité doit être préservée (phénotype souche, capacité de différenciation). La mise en culture de ces cellules, sur des microporteurs, en bioréacteur agité, semble répondre à ces enjeux. Cependant, une connaissance plus précise de l’impact, sur la réponse physiologique des cellules, des technologies utilisées et de l’hydrodynamique générée est nécessaire pour améliorer les lois d’extrapolation des bioréacteurs de culture de CSM. Dans ce contexte, des travaux ont été mis en œuvre pour étudier l’influence du mode d’agitation (orbital ou mécanique) sur l’attachement, l’expansion et le détachement de CSM issues de la gelée de Wharton (GW-CSM) de cordons ombilicaux, sur des microporteurs de différentes compositions. Pour contribuer à la quantification de l’expansion cellulaire, une méthode de comptage automatique in situ a été développée pour estimer le nombre de cellules par microporteur, ainsi que leur répartition, sans avoir à procéder à leur détachement. Des microporteurs commerciaux ont ensuite pu être comparés à des microporteurs synthétisés dans un laboratoire partenaire, en termes d’attachement et expansion cellulaire, ainsi que de facilité de détachement. En parallèle de ces travaux, l’impact de la conception du mobile d’agitation, en bioréacteur mécaniquement agité, sur la mise en suspension de microporteurs a été analysé. A l’issue de cette étude, une analyse dimensionnelle et des simulations CFD ont été mises en place et deux modèles reliant la fréquence minimale de juste mise en suspension (Njs) avec la géométrie du mobile d’agitation (forme, taille, position dans la cuve) et les propriétés matérielles des particules et de la phase liquide ont été proposés. Une stratégie d’optimisation des paramètres géométriques d’un mobile en minibioréacteur, dédié à la culture de CSM sur microporteurs, a été mise en place, à partir de paramètres caractérisant les contraintes hydromécaniques perçues par la phase solide, judicieusement choisis et intégrés lors des simulations CFD. Selon un plan d’expérience, et les résultats extraits des simulations, des surfaces de réponse ont été construites et une optimisation multi-objective a été réalisée afin de déterminer la géométrie minimisant les contraintes perçues par les particules, et donc par les cellules adhérées. Des cultures de GW-CSM en minibioréacteurs équipés de différents mobiles ont finalement été validées, avec une comparaison préliminaire de l’impact de ces géométries sur l’expansion cellulaire<br>Mesenchymal stem cells (MSC) are becoming increasingly involved in the regenerative medicine field, particularly to treat diseases that are not effectively curable with the current therapies. Two scientific barriers are nevertheless responsible for MSC use and commercialization limitations. On one side, large amounts of cells are needed to reach the high cell dose requirements. On the other side, cells being the final product themselves, directly injected into the patient, their quality have to be controlled (stem cell phenotype, differentiation capability). MSC cultivation on microcarriers in a stirred bioreactor seems to meet these challenges. However, a precise knowledge about the impact of the technologies and the hydrodynamics generated, on the physiological cell response, is necessary to improve the scale-up of MSC cultures in bioreactors. In this context, present work is dedicated to the study of the impact of the agitation mode (orbital or mechanical) on the cell attachment, expansion and detachment on various microcarrier types, in the case of MSC derived from the Wharton’s jelly (WJ-MSC) of umbilical cords. To quantify more precisely cell distribution and expansion on microcarriers, an automatic and in situ counting method was developed, which need no detachment step. This allowed the identification of commercial microcarriers suitable for WJ-MSC cultures, which were then compared to home-made microcarriers, synthesized by a partner laboratory, in terms of cell attachment and expansion, and detachment efficiency. In parallel to these works, the impact of the impeller design on the microcarrier suspension in stirred tank bioreactors was investigated. Based on a dimensional analysis and CFD simulations, it resulted in the establishment of two models relating the minimal agitation rate to ensure all particle suspension (Njs) with the impeller geometrical characteristics (design, size, off-bottom clearance) and the material properties of both the solid and the liquid phases. CFD models validation allowed then to develop a strategy to optimize the geometrical configuration of an impeller, dedicated to MSC cultures on microcarriers in a minibioreactor. Parameters characterizing the hydromechanical stress encountered by the solid phase were wisely chosen and integrated into CFD simulations. Based on a design of experiments, and the hydrodynamics data recovered from simulations, response surfaces were built and a multiobjective optimization was achieved in order to determine the geometry minimizing the particle stress, and also by adhered cells. WJ-MSC cultures in minibioreactors equipped with impellers displaying various geometries were finally validated, with a preliminary comparison of the impact of these geometries on the cell expansion

A cartilagem articular é um tecido avascular, com baixa celularidade, composta principalmente de colágeno extracelular e proteoglicanos, com uma capacidade limitada de regeneração. Nos últimos anos, diversas abordagens clínicas e de pesquisa têm sido adotadas para reparar danos na cartilagem articular, como transplante de condrócitos, enxerto de periósteo, células-tronco mesenquimais e tecidos derivados dessas células. O isolamento de células-tronco mesenquimais foi relatado a partir de diferentes tecidos, incluindo medula óssea, tecido adiposo, sangue do cordão umbilical, sangue periférico e saco vitelino em equinos. As células-tronco mesenquimais derivadas de líquido e membrana sinoviais foram obtidas em humanos, cães, suínos e caprinos e são fontes promissoras para regeneração articular, já que são tecido-específicas e de fácil obtenção e cultivo. O objetivo deste trabalho foi estabelecer e caracterizar a linhagens de células obtidas de membrana e líquido sinoviais equinos. Os fragmentos foram obtidos por meio de artroscopias e cultivados em meios de cultura DMEM-H e MEM para obtenção das linhagens. Para análise da morfologia celular foi realizada a fotodocumentação das garrafas em microscopia invertida. A expressão de marcadores de células-tronco (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 e Ly6a), marcadores inflamatórios (COX-2, TNF-R1-r, CD11, CD1a e MCP-1) e marcadores envolvidos na checagem e progressão do ciclo celular (Caspase-3 fosforilada, HSP-47, P21, Ki67, Ciclina D1 e P53) mostraram diferencialmente expressos, sugerindo que podemos considerá-las uma possível fonte de células-tronco mesenquimais. Devido ao grande impacto que patologias na articulação têm sobre o desempenho atlético em cavalos, os resultados demonstrados neste trabalho será uma base para conduzir outros experimentos na avaliação das aplicações terapêuticas dessas células.<br>The articular cartilage is an avascular tissue with low cellularity composed of extracellular collagen and proteoglicans. It has a limited capacity of regeneration. Condrocyte transplantation, mesenchymal stem cells and tissues derived from these cells has been used by several researches to repair damage to the articular cartilage. The isolation of mesenchymal stem cells has been reported from different tissues such as bone marrow, adipose tissue, blood, umbilical cord, and yolk sac. The mesenchymal stem cells from synovial fluid and synovial membrane were obtained from humans, dogs, pigs and goats. These cells are tissue specific and easy to obtain and cultivate. The objective of this research is to obtain and characterize cells from equine synovial fluid and synovial membrane. The samples were obtained by arthroscopy and cultivated in the DMEM-H and MEM media. Cell morphology analyses were made by photodocumentation in inverted microscopy. The expression of stem cell markers (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 and Ly6a), inflammation markers (COX-2, TNF-R1-r, CD11, CD1a and MCP-1) and markers involved in checking and cell cycle progression (Caspase-3, HSP-47, P21, Ki67, Ciclina D1 and P53) showed differentially expressed. Mesenchymal stem cells from synovial membrane and synovial fluid provide promise for cell-based therapies for articular cartilage repair. These results may lead other experiments to use these cells to new therapeutic applications.

Les cellules souches mésenchymateuses (CSM) interviennent de plus en plus dans le domaine de la médecine régénérative, notamment pour traiter des maladies aujourd’hui difficilement curables avec les moyens actuels. Deux verrous scientifiques limitent pourtant leur utilisation et leur commercialisation. D’une part, de grandes quantités de cellules sont nécessaires pour répondre à la forte demande médicale. D’autre part, les cellules étant elles-mêmes le médicament final, délivré chez le patient, leur qualité doit être préservée (phénotype souche, capacité de différenciation). La mise en culture de ces cellules, sur des microporteurs, en bioréacteur agité, semble répondre à ces enjeux. Cependant, une connaissance plus précise de l’impact, sur la réponse physiologique des cellules, des technologies utilisées et de l’hydrodynamique générée est nécessaire pour améliorer les lois d’extrapolation des bioréacteurs de culture de CSM. Dans ce contexte, des travaux ont été mis en œuvre pour étudier l’influence du mode d’agitation (orbital ou mécanique) sur l’attachement, l’expansion et le détachement de CSM issues de la gelée de Wharton (GW-CSM) de cordons ombilicaux, sur des microporteurs de différentes compositions. Pour contribuer à la quantification de l’expansion cellulaire, une méthode de comptage automatique in situ a été développée pour estimer le nombre de cellules par microporteur, ainsi que leur répartition, sans avoir à procéder à leur détachement. Des microporteurs commerciaux ont ensuite pu être comparés à des microporteurs synthétisés dans un laboratoire partenaire, en termes d’attachement et expansion cellulaire, ainsi que de facilité de détachement. En parallèle de ces travaux, l’impact de la conception du mobile d’agitation, en bioréacteur mécaniquement agité, sur la mise en suspension de microporteurs a été analysé. A l’issue de cette étude, une analyse dimensionnelle et des simulations CFD ont été mises en place et deux modèles reliant la fréquence minimale de juste mise en suspension (Njs) avec la géométrie du mobile d’agitation (forme, taille, position dans la cuve) et les propriétés matérielles des particules et de la phase liquide ont été proposés. Une stratégie d’optimisation des paramètres géométriques d’un mobile en minibioréacteur, dédié à la culture de CSM sur microporteurs, a été mise en place, à partir de paramètres caractérisant les contraintes hydromécaniques perçues par la phase solide, judicieusement choisis et intégrés lors des simulations CFD. Selon un plan d’expérience, et les résultats extraits des simulations, des surfaces de réponse ont été construites et une optimisation multi-objective a été réalisée afin de déterminer la géométrie minimisant les contraintes perçues par les particules, et donc par les cellules adhérées. Des cultures de GW-CSM en minibioréacteurs équipés de différents mobiles ont finalement été validées, avec une comparaison préliminaire de l’impact de ces géométries sur l’expansion cellulaire<br>Mesenchymal stem cells (MSC) are becoming increasingly involved in the regenerative medicine field, particularly to treat diseases that are not effectively curable with the current therapies. Two scientific barriers are nevertheless responsible for MSC use and commercialization limitations. On one side, large amounts of cells are needed to reach the high cell dose requirements. On the other side, cells being the final product themselves, directly injected into the patient, their quality have to be controlled (stem cell phenotype, differentiation capability). MSC cultivation on microcarriers in a stirred bioreactor seems to meet these challenges. However, a precise knowledge about the impact of the technologies and the hydrodynamics generated, on the physiological cell response, is necessary to improve the scale-up of MSC cultures in bioreactors. In this context, present work is dedicated to the study of the impact of the agitation mode (orbital or mechanical) on the cell attachment, expansion and detachment on various microcarrier types, in the case of MSC derived from the Wharton’s jelly (WJ-MSC) of umbilical cords. To quantify more precisely cell distribution and expansion on microcarriers, an automatic and in situ counting method was developed, which need no detachment step. This allowed the identification of commercial microcarriers suitable for WJ-MSC cultures, which were then compared to home-made microcarriers, synthesized by a partner laboratory, in terms of cell attachment and expansion, and detachment efficiency. In parallel to these works, the impact of the impeller design on the microcarrier suspension in stirred tank bioreactors was investigated. Based on a dimensional analysis and CFD simulations, it resulted in the establishment of two models relating the minimal agitation rate to ensure all particle suspension (Njs) with the impeller geometrical characteristics (design, size, off-bottom clearance) and the material properties of both the solid and the liquid phases. CFD models validation allowed then to develop a strategy to optimize the geometrical configuration of an impeller, dedicated to MSC cultures on microcarriers in a minibioreactor. Parameters characterizing the hydromechanical stress encountered by the solid phase were wisely chosen and integrated into CFD simulations. Based on a design of experiments, and the hydrodynamics data recovered from simulations, response surfaces were built and a multiobjective optimization was achieved in order to determine the geometry minimizing the particle stress, and also by adhered cells. WJ-MSC cultures in minibioreactors equipped with impellers displaying various geometries were finally validated, with a preliminary comparison of the impact of these geometries on the cell expansion

As células-tronco mesenquimais (CTM) são células progenitoras indiferenciadas que apresentam altas taxas de proliferação em cultivo, capacidade de diferenciação em inúmeros tecidos e podem ser encontradas no organismo adulto e, também, em tecidos fetais, como cordão umbilical, placenta e liquido amniótico (LA). Estudos demonstraram que o LA humano obtido por amniocentese no segundo trimestre de gestação, comumente utilizado em exames de diagnóstico fetal, apresenta-se como uma fonte em potencial destas células progenitoras. Contudo, estas células necessitam de mais estudos quanto às técnicas de isolamento, expansão e, sobretudo, acerca dos protocolos de congelamento utilizados em sua criopreservação. Com isso, nos propusemos a padronizar técnicas de cultivo para as CTLA, como melhor meio de cultura e densidade de inóculo; avaliar as características biológicas como estado de indiferenciação, ciclo celular, marcadores de membrana, plasticidade e, ainda, testar dois protocolos de congelamento celular (padrão e gradual) com diferentes criopreservantes (DMSO, glicerol, trealose e sacarose) que mantivessem uma alta viabilidade e as demais características das células-tronco após períodos de 3 e 6 meses de armazenamento em nitrogênio líquido. Ao fim dos experimentos constatamos ser o líquido amniótico uma rica fonte de CTM passíveis de serem isoladas e cultivadas com meio de cultura a-MEM suplementado com 20% de SFB. Padronizamos, também, uma contagem celular inicial das amostras para otimizar o plaqueamento primário, uma densidade de inóculo ideal para as passagens posteriores (5.000 céls/cm2) e o tempo de dobramento (30 ± 4 horas) das mesmas. As CTLA expressaram genes de indiferenciação: Oct-4, Sox-2 e Nanog; apresentaram positividade para marcadores de superfície CD29, CD44, CD90 e CD105; alta taxa de proliferação in vitro e diferenciaram-se em tecido ósseo, adiposo, cartilaginoso e neuronal. Não encontramos diferenças significativas entre os dois métodos de congelamento avaliados no que diz respeito à viabilidade pós-congelamento. Todos os criopreservantes analisados mantiveram o estado de indiferenciação e plasticidade das células-tronco congeladas por 3 e 6 meses, contudo o DMSO 10% proporcionou maiores taxas de viabilidade do que os demais. As CTLA ficam desta maneira melhor caracterizadas, com protocolos de cultivo e estocagem bem estabelecidos, facilitando a produção de células-tronco funcionais em larga escala aptas a serem utilizadas em experimentos futuros<br>Mesenchymal stem cells (MSCs) are undifferentiated progenitor cells that have high proliferation rates in culture, ability to differentiate into various tissues and can be found in adult and fetal tissues such as umbilical cord, placenta and amniotic fluid (AF). Studies showed that human AF obtained by amniocentesis in second trimester of pregnancy, commonly used in fetal diagnostic, is a potential source of progenitor cells. However, these cells require further studies above techniques of isolation, expansion and, especially, about freezing protocols used in their cryopreservation. For then, we analyzed isolation and expansion methods to AFSC as the best culture medium and inoculum density; biological characteristics such as undifferentiated state, cell cycle, membrane markers, plasticity, and also we tested two freezing protocols (standard and graduated) with different cryoprotectors (DMSO, glycerol, trehalose and sucrose) that could be able to maintain high viability and other characteristics of stem cells after 3 and 6 months of storage in liquid nitrogen. At the end of experiments we found that amniotic fluid is a rich source of mesenchymal stem cells that can be isolated and cultured with a-MEM medium supplemented with 20% FBS. Standardized an initial cell samples counting to optimize primary plating, an ideal inoculum density for the later passages (5000 cells/cm2) and doubling time (30 ± 4 hours). AFSC expressed undifferentiated genes: Oct-4, Sox-2 and Nanog, were positive for surface markers CD29, CD44, CD90 and CD105; presented high in vitro proliferation rate and capability to differentiate into bone, adipose, cartilage and neuronal tissues. There was not statistical significance in cell viability between standard and graduated freezing protocols. All evaluated cryoprotectors maintained the basic features of amniotic fluid stem cells, as Oct-4 gene expression, surface markers and plasticity. DMSO 10% showed higher rates of viability than the others. We can conclude that AF is a rich source of MSC with great capacity for expansion and differentiation and that both methods of freezing could be used for AF cells storage. All tested cryoprotectors maintains stemness of AFSC, therefore the highest viability rate is supplied by 10% DMSO. In this way, AFSC are better characterized, with cultivation and storage protocols standardized, resulting in large scale production of functional stem cells suitable for use in future experiments

Na espécie equina, as enfermidades osteoarticulares causam prejuízo econômico e impacto negativo no desempenho atlético, devido aos danos causados na cartilagem articular. A regeneração da cartilagem hialina e a manutenção da integridade das estruturas que a compõe norteiam a busca do tratamento ideal. Neste contexto, este estudo foi delineado com o objetivo de investigar a presença de células-tronco mesenquimais (CTMs) no líquido sinovial (LS) e na membrana sinovial (MS) de equinos com articulações hígidas, com osteocondrite dissecante (OCD) e com osteoartrite (OA) e compará-las, visando estabelecer qual fonte celular possui melhor característica fenotípica e capacidade de diferenciação celular, mais especificamente, aquela que seja superior em relação à capacidade condrogênica. Foram utilizados equinos machos e fêmeas de diferentes idades, totalizando 97 articulações. O LS e MS foram coletados durante artroscopia e as células foram cultivadas, e avaliadas por citometria de fluxo com os anticorpos CD44, CD90, CD105, CD34; e por imunocitoquímica com os anticorpos nanog, oct4, PGP 9.5, lisozima, vimentina e citoqueratina. Adicionalmente, o potencial de diferenciação das células foi avaliado para as linhagens condrogênica, osteogênica e adipogênica. Foi realizado teste de tumorigenicidade em camundongos Balb-Cnu/nu, para comprovar aplicabilidade clínica, e posteriormente, as CTMs provenientes de LS de articulações hígidas foram aplicadas em articulações de equinos. A identidade das células foi comprovada durante o cultivo demonstrando características de adesão ao plástico e morfologia fibroblastóide. A média percentual das populações positivas para CD90 foi de 64,9% (LS-H), 48,3% (LS-OCD), 48,1% (LS-OA), 66,6% (MS-H), 40,2% (MS-OCD) e 40,3% (MS-OA). A porcentagem de células positivas para CD44 foi de 1,18% (LS-H), 3,98% (LS-OCD), 14,2% (LS-OA), 1,9% (MS-H), 2,17% (MS-OCD) 8,56% (MS-OA). Não foi observada expressão dos anticorpos CD34 e CD105. Na análise imunocitoquímica foi detectada expressão positiva para os anticorpos: lisozima, PGP 9.5, PCNA e vimentina, e negativa para nanog, oct4 e citoqueratina. A multipotência (osteogênica, condrogênica e adipogênica) das células foi confirmada através da coloração Alizarin Red para detecção de matriz de cálcio, Oil Red O para detecção de gotículas de gordura e azul de toluidina, alcian blue e hematoxilina eosina para detecção de matriz de proteoglicanos. Com relação aos resultados do teste tumorigênico, nenhum órgão dos camundongos foi afetado, assegurando a aplicabilidade das células estudadas. Ainda, as articulações de equinos tratadas, não apresentaram quaisquer sinais de reação inflamatória após aplicação de células alogênicas. Por fim, concluímos que, a fenotipagem positiva de CD44 e CD90 somada à capacidade de diferenciação nas linhagens osteogênica e condrogênica confirma a presença de CTMs nas populações celulares obtidas de LS e MS de equinos. Também foi observado que as células de LS provenientes de articulações hígidas, são as de melhor utilização clínica, uma vez que apresentaram maior expressão de CD90 e demonstraram melhor capacidade de diferenciação celular em relação às células derivadas de articulações enfermas. Além disso, possuem método mais fácil de colheita em relação à colheita de MS, visando futura terapia celular na rotina clínica<br>In the equine species, osteoarticular diseases cause significant economic losses and negative impact on equine athletic performance. The hyaline cartilage regeneration and the maintenance of integrity of its components guide the search for the ideal treatment. In this scenario, this study aimed to investigate the presence of mesenchymal stem cell (MSCs) in the synovial fluid (SF) and in the synovial membrane (SM) of healthy equine joints, osteoarthritic (OA) and osteochondritic joints (OCD), comparing their potential as cellular sources, according to their differentiation ability, in particular with superior chondrogenic potential and the phenotypic characteristics of the MSCs. Ninety-seven equine joints from males and females of different ages were used to harvest cells. SF and SM were obtained during arthroscopy and the cells SF and SM were cultured and assessed for CD90, CD44, CD105 and CD34 markers by flow cytometry, and nanog, oct4, PGP 9.5, lyzozyme, vimentin and cytokeratin were assessed by immunocytochemistry. Additionally, cells were evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. The tumorigenicity test was carried in Balb-C nu/nu mice, to verify the safety of cell sources and, later, mesenchymal stem cells harvested from healthy equine joints were injected into equine joints. The identity of these cells was confirmed during cell growth, through properties of plastic adhesion and fibroblastoid morphology. The mean percentage of CD90 positive cells was 64.9% (SF-H), 48.3% (SF-OCD), 48.1% (SF-OA), 66.6% (SM-H), 40.2% (SM- OCD) and 40.3% (SM-OA). The percentage of CD44 positive cells was 1.18 % (SF-H), 3.98% (SF-OCD), 14.2% (SF-OA), 1.9% (SM-H), 2.17% (SM-OCD) and 8.56% (SM-OA). The expression of CD34 and CD105 antibodies was not observed. Through immunocytochemical analysis, expression for lysozyme, PGP9.5, PCNA e vimentin antibodies was detected and negative expression for nanog, oct4 e cytokeratin was observed. The multipotent capacity of mesenchymal stromal cells for lineage differentiation (osteogenic, chondrogenoic and adipogenic) was confirmed with different staining techniques: Alizarin Red enabled detection of the calcium matrix, Oil Red O enabled the detection of fat droplets and Toluidin Blue, Alcian Blue and haematoxylin eosin enabled detection of proteoglycan matrix. Results of tumorigenic tests in mice showed no compromise of any internal organ, assuring applicability of the studied cells. Furthermore, equine joints treated with MSC harvested from healthy joints did not show any signs of an inflammatory reaction after injection of the allogeneic cells. The presence of cells with positive CD44 and CD90 phenotypes and with the ability to differentiate into osteogenic and chondrogenic lineages confirms the presence of MSCs in equine SF and SM. Cells obtained from healthy SF were more suitable for clinical application, for they presented higher CD90 expression and demonstrated greater differentiation capabilities, when compared to that of cells retrieved from compromised joints. In addition to that, SF derived cells are easier to obtain when compared to SM cells, aiming their future application clinical

Malformações fetais são importantes causas de óbito fetal e mortalidade infantil. A medicina regenerativa apresenta-se como a terceira modalidade terapêutica fetal. Células obtidas do líquido amniótico mostram-se como opção preferencial em terapia celular por sua alta taxa de proliferação in vitro, habilidade de autorrenovação e potencial multilinhagem. Para uso clínico, é recomendável a exclusão de material animal não humano do meio de cultura dessas células, prevenindo reações imunes, transmissão de príons, bactérias e vírus. O soro fetal bovino é componente usual do meio de cultura dessas células. Sua substituição por soro humano previne essas possíveis consequências. O objetivo deste trabalho foi estudar células obtidas do líquido amniótico, em meio de cultura suplementado por soro fetal bovino ou humano e compará-las. Foram avaliadas seu isolamento e cultivo, padrão de crescimento, morfologia, imunofenótipo, capacidade de diferenciação osteogênica e condrogênica, e caracterizou-se a expressão dos genes OCT-4, NANOG e SOX2. Foram analisadas amostras provenientes de cinco gestantes, obtidas por amniocentese de segundo trimestre, indicadas por idade materna avançada. As células do líquido amniótico, após centrifugação, foram colocadas em frasco de cultura com meio -MEM suplementado com 20% de soro fetal bovino (grupo B) ou soro humano (grupo H). Ao atingirem 70% de confluência, foram tripsinizadas e expandidas. Após a terceira passagem celular foram separadas alíquotas do grupo B e H para avaliação de seu potencial de expansão, por meio da construção de curvas de crescimento celular para diferentes inóculos iniciais (1.000, 5.000, 10.000 e 15.000 células). Sua morfologia foi avaliada por microscopia ótica através da coloração de Leishman e por microscopia eletrônica. A análise do imunofenótipo das células dos dois grupos foi realizada por citometria de fluxo, analisando-se os marcadores de superfície: CD14, CD29, CD31, CD34, CD44, CD45, CD90, CD105, CD106, CD117 e CD133. A diferenciação osteogênica foi realizada pelo acréscimo ao meio de cultura de fosfato-2-ascorbato e -glicerofosfato, comprovada pela produção de cálcio pelas células, coradas pela Alizarina e visualização da Fosfatase Alcalina nas células. A diferenciação condrogênica foi realizada pelo acréscimo ao meio de cultura de dexametasona e TGF-1, comprovada pela análise histológica após coloração pela hematoxilina-eosina. Avaliou-se a expressão gênica dos transcritos OCT-4, NANOG e SOX2, por RT-PCR das células dos dois grupos (B e H), comparadas aos controles celulares MRC-5 e NTERA-2 cl.D1. Observou-se alta taxa de expansão, sem diferença significativa entre os grupos (p=0,715), mesmo considerando-se a evolução dia-a-dia (p=0,681) e a alta viabilidade celular, com média de 94% nos dois grupos (p=0,686). A análise morfológica das células dos dois grupos revelou aspecto mesenquimal típico, com crescimento celular em cultura também típico dessa linhagem. Ao microscópio eletrônico, observou-se maior número de vacúolos lipídicos naquelas células do grupo H. A imunofenotipagem demonstrou marcação positiva para linhagem mesenquimal e negativa para outras linhagens. Ocorreu diferenciação osteogênica e condrogênica e expressão dos transcritos OCT-4, NANOG e SOX2. Não houve diferença significativa nos aspectos estudados, entre os grupos B e H. Concluiu-se que essas células, isoladas do líquido amniótico, apresentam característica de fácil isolamento, alta taxa de proliferação, aspecto morfológico e imunofenótipo mesenquimal, capacidade de diferenciação osteogênica e condrogênica, expressando os transcritos OCT-4, NANOG e SOX2, associados à pluripotência celular, tanto em meio suplementado por soro fetal bovino como por soro humano.<br>Fetal malformations are a significant cause of fetal death and infant mortality. Regenerative medicine is presented as a third therapeutic method. Cells obtained from the amniotic fluid are seen to be an option of choice for cell therapy because of their high rate of in vitro proliferation, power of selfrenewal and multi-lineage potential. For clinical use the exclusion of nonhuman animal material from the culture medium of these cells is to be recommended, thus precluding immune reactions and the transmission of prions, bacteria and viruses. Bovine fetal serum is a normal component of the culture medium of these cells. Its replacement by human serum precludes those possible consequences. The objective of this present study was investigation into the cells obtained from the amniotic fluid, in a culture medium supplemented with bovine fetal or human serum, and compare them. Their isolation and culture, growth rate, morphology, immune phenotype and osteogenic and chondrogenic capacity were assessed and the expression of the OCT-4, NANOG and SOX2 genes was characterized. Samples taken from five pregnant women by amniocentesis during the second trimester, recommended by virtue of advanced maternal age, were analyzed. The cells of the amniotic fluid, after centrifugation, were placed in a culture flask with an -MEM medium supplemented with 20% of bovine fetal serum (group B) or human serum (group H). After attaining 70% confluence they were trypsinized and expanded. After the third cellular passage they were separated into quotas of groups B and H for assessment of their expansion potential, by means of the construction of cellular growth curves for the different initial inocula (1,000, 5,000, 10,000 and 15,000 cells). Their morphology was assessed by optical microscopy by means of Leishman´s staining and electron microscopy. The analysis of the immune phenotype of the cells of the two groups was undertaken by flow cytometry, the surface markers CD14, CD29, CD31, CD34, CD44, CD45, CD90, CD105, CD106, CD117 and CD133 being analyzed. The osteogenic differentiation was undertaken by the addition of ascorbate-2-phosphate-2 and - glycerophosphate to the culture medium and proved by the production of calcium by the cells, stained with Alizarin, and visualization of the Alkaline Phosphatase in the cells. The chondrogenic differentiation was brought about by the addition of dexamethasone and TGF-1 to the culture medium and demonstrated by histological analysis after staining with hematoxilineeosine. The genic expression of the OCT-4, NANOG and SOX2 transcripts was compared with the control cells MRC-5 and NTERA-2 cl.D1 by the RTPCR of the cells of the two groups (B and H). A high expansion rate was observed, with no significant difference between the groups (p=0.715), even when the day-to-day progress (p=0.681) was taken into consideration, and high cell viability, with an average of 94% in the two groups (p=0.686). The morphological analysis of the cells of the two groups presented a typical mesenchymal aspect, with cell growth in the culture also typical of this line. Under the electron microscope, a greater number of lipid vacuoles were observed in the cells of the H group. The immune phenotyping presented a positive marking for the mesenchymal line but a negative one for the others. Osteogenic and chondrogenic differentiation occurred as also did expression of the transcripts OCT-4, NANOG and SOX2. There was no significant difference, as regards the aspects studied, between groups B and H. It is concluded that these cells isolated from the amniotic fluid were characterized by ease of isolation, a high rate of proliferation, mesenchymal morphological aspect and immune phenotype, capacity for osteogenic and chondrogenic differentiation, expressing the transcripts OCT-4, NANOG and SOX2, both in the medium supplemented with bovine fetal serum and in that with human serum.

Células tronco mesenquimais (CTm) apresentam potencial para tratamento da doença renal pela possibilidade de promover regeneração tecidual e recuperação funcional, possivelmente por seus efeitos parácrinos. Na última década, o líquido amniótico foi descrito como uma fonte promissora de extração e isolamento de CTm. Alguns estudos mostraram o efeito renoprotetor das CTm derivadas do líquido amniótico (CTmLA) na doença renal aguda e crônica, quando inoculadas precocemente. Entretanto, ainda não foi estudado o efeito da administração de CTmLA em modelo experimental de doença renal crônica (DRC) com a lesão já estabelecida, situação esta que reproduz melhor a apresentação clínica da doença nos pacientes. Assim, o objetivo do presente estudo foi analisar o efeito da inoculação de CTmLA na região subcapsular renal no modelo de DRC já estabelecido. As CTmLA foram obtidas de pacientes no segundo trimestre de gestação e isoladas através da sua capacidade de aderência ao plástico. A caracterização das CTm foi feita por citometria de fluxo e pela diferenciação celular in vitro. O modelo de DRC utilizado foi o de nefrectomia 5/6 (Nx) que, pela perda de massa renal, evolui com hipertensão arterial, proteinúria, glomeruloesclerose, fibrose intersticial e perda progressiva da função renal. Quinze dias após a indução do modelo, estas alterações já são marcantes e agravam-se com 30 dias. Foram realizados 2 protocolos experimentais: no protocolo I, os animais Nx com DRC estabelecida receberam dose única de CTmLA (5x105) na região subcapsular renal e foram acompanhados por 30 e 60 dias de experimento. No protocolo II, os animais Nx com DRC estabelecida receberam duas doses de CTmLA (5x105) na região subcapsular renal, no 15° e 30° dia após a nefrectomia 5/6, e foram acompanhados por 30 dias, totalizando 60 dias de experimento. Os animais foram subdivididos nos grupos: Sham, ratos submetidos à cirurgia fictícia; Sham+CTmLA, ratos submetidos à Sham que receberam CTmLA; Nx, ratos submetidos à nefrectomia 5/6; Nx+CTmLA, ratos Nx que receberam CTmLA. Para verificar a localização das CTmLA no tecido renal foi realizada a hibridização in situ para cromossomo XY. Foram realizadas análises dos parâmetros clínicos e laboratoriais, além de análise histológica, imunohistoquímica, PCR em tempo real e multiplex. Resultados: as CTmLA cultivadas mostraram grande capacidade de aderência, crescimento em colônia e de diferenciação em células osteogênicas, adipogênicas e condrogênicas. A análise por citometria mostrou-se positiva para CD29, CD44, CD90 e CD105, com uma pequena população de células de CD14, CD34, CD45 e CD117, confirmando a presença preponderante de CTm. Protocolo I: Após 30 dias, a inoculação de CTmLA, dose única, preveniu a elevação da pressão arterial, da proteinúria, da glomeruloesclerose, recuperando a expressão dos marcadores de podócitos, WT-1 e sinaptopodina. Entretanto, não houve efeito benéfico nos níveis de creatinina sérica e na fibrose intersticial, após 30 e 60 dias. O tratamento com CTmLA promoveu uma diminuição marcante do número de macrófagos e uma discreta queda dos leucócitos no infiltrado inflamatório renal, além da diminuição do número de miofibroblastos no interstício renal. Citocinas pró-inflamatórias foram encontradas em menor concentração no tecido renal dos animais que receberam CTmLA (IL-1beta, TNF-alfa, MCP-1 e RANTES). Não houve alteração significativa das citocinas Th1 e Th2, exceto por um aumento da IL-4 nos animais tratados com CTmLA. Os animais que foram acompanhados por 60 dias tiveram uma melhora da proteinúria, da glomeruloesclerose, diminuição do infiltrado de macrófagos e uma melhora da expressão de WT-1. Não foram observadas diferenças estatísticas nos parâmetros de creatinina sérica e fibrose intersticial, aos 30 e 60 dias. Protocolo II: Nos animais que receberam a segunda dose de CTmLA e foram acompanhados por 60 dias observou-se prevenção da elevação da pressão arterial e da proteinúria, além de uma marcante diminuição da fibrose intersticial. Em conclusão, o presente estudo mostrou, pela primeira vez, que a terapia com CTmLA foi capaz de induzir renoproteção nos animais com doença renal crônica estabelecida. O tratamento com CTmLA pode representar uma nova abordagem terapêutica bloqueando a progressão da doença renal crônica<br>Mesenchymal stem cells (mSC) represent therapeutic potential for the treatment of renal diseases, due to their ability to induce tissue regeneration and functional recovery. Human amniotic fluid stem cells (AFmSC) are a class of fetal, pluripotent stem cells, which present characteristics intermediate between embryonic and adult stem cells. These cells are characterized by the expression of mesenchymal stem cells markers. In addition, they have the ability to differentiate into lineages of all embryonic germ layers. They also show high proliferative rates, but do not induce tumor formation. Therefore, AFmSC are considered to be a very promising cell source and these characteristics have generated a great interest concerning their potential renoprotective effects. The aim of this study was to analyze the effects of AFmSC in an experimental model of chronic kidney disease, the 5/6 nephrectomy model (Nx), after the disease has been established, in order to more closely resemble the clinical settings in humans. AFmSC derived from second-trimester amniocentesis were isolated by plastic adhesion. After 4-7 passages, AFmSC characteristics were confirmed by flow cytometry and by their ability to differentiate into osteogenic, adipogenic and chondrogenic lineages. Two experimental protocols were performed: In protocol I, rats underwent 5/6 nephrectomy (Nx) or sham surgery at day 0, received at day 15 a single dose of hAFmSC (5x105 cells) injected under the renal capsule and were studied at day 30 and 60 days. In protocol II, rats underwent Nx or sham surgery, and received at days 15 and 30, two doses of hAFmSC (5x105 cells) injected under the renal capsule, and were studied at day 60. In both protocols, the animals were subdivided into four groups: Sham, rats submitted to fictitious surgery; Sham+hAFmSC, Sham rats that received hAFmSC; Nx, rats submitted to nephrectomy 5/6; Nx+hAFmSC, Nx rats receiving hAFmSC. The hAFmSC were followed in the renal tissue by in situ hybridization for XY chromosome. In all the groups, clinical and histological parameters were analyzed by immunohistochemistry and real-time PCR. Results: AFmSC cultivated demonstrated an ability to adhere to plastic, to grow in colonies and to differentiate in osteogenic, adipogenic and chondrogenic cells. Quantitative analysis of cell markers by flow cytometry showed that isolated cells were positive for CD29, CD44, CD90 and CD105, with a small population of cells positive for CD14, CD34, CD45 and CD117, confirming a preponderant presence of mSC. Protocol I: After 30 days, the single dose of hAFmSC significantly reduced the blood pressure levels, proteinuria, glomerulosclerosis and improved the expression of podocytes markers, WT-1 and synaptopodin. A marked decrease on the number of macrophages and a discrete decrease of leucocyte infiltration, as well as a reduction of interstitial myofibroblasts was observed. Treatment with hAFmSC significantly reduced some proinflammatory cytokines (IL1beta, TNF-alpha, MCP-1 and RANTES). No significant difference in Th1 or Th2 cytokines was observed, except for IL-4 increase in Nx rats treated with hAFmSC. At 60 days of follow-up, Nx rats treated with hAFmSC presented reduced proteinuria, glomerulosclerosis and macrophages besides increase in WT-1 expression. No improvements were observed on serum creatinine and of interstitial fibrosis, after 30 and 60 days. Protocol II: Inoculation of two doses of hAFmSC in Nx rats improved blood pressure levels, proteinuria and interstitial fibrosis at day 60. In conclusion, the present study demonstrated, for the first time, that hAFmSC induced renoprotection in animals with established chronic kidney disease. Treatment with hAFmSC may represent a novel therapeutic approach for blocking the progression of chronic kidney disease

The following thesis presents a computational framework that can capture inherently non-linear and emergent biocomplex phenomena. The main motivation behind the investigations undertaken was the absence of a suitable platform that can simulate, both the continuous features as well as the discrete, interaction-based dynamics of a given biological system, or in short, dynamic reciprocity. In order to determine the most powerful approach to achieve this, the efficacy of two modelling paradigms, transport phenomena as well as agent-based, was evaluated and eventually combined. Computational Fluid Dynamics (CFD) was utilised to investigate optimal boundary conditions, in terms of meeting cellular glucose consumption requirements and exposure to physiologically relevant shear fields, that would support mesenchymal stem cell growth in a 3-dimensional culture maintained in a commercially available bioreactor. In addition to validating the default bioreactor configuration and operational parameter ranges as suitable towards sustaining stem cell growth, the investigation underscored the effectiveness of CFD as a design tool. However, due to the homogeneity assumption, an untenable assumption for most biological systems, CFD often encounters difficulties in simulating the interaction-reliant evolution of cellular systems. Therefore, the efficacy of the agent-based approach was evaluated by simulating a morphogenetic event: development of in vitro osteogenic nodule. The novel model replicated most aspects observed in vitro, which included: spatial arrangement of relevant players inside the nodule, interaction-based development of the osteogenic nodules, and the dependence of nodule growth on its size. The model was subsequently applied to interrogate the various competing hypotheses on this process and identify the one that best captures transformation of osteoblasts into osteocytes, a subject of great conjecture. The results from this investigation annulled one of the competing hypotheses, which purported the slow-down in the rate of matrix deposition by certain osteoblasts, and also suggested the acquisition of polarity to be a non-random event. The agent-based model, however, due to being inherently computationally expensive, cannot be recommended to model bulk phenomena. Therefore, the two approaches were integrated to create a modelling platform that was utilised to capture dynamic reciprocity in a bioreactor. As a part of this investigation, an amended definition of dynamic reciprocity and its computational analogue, dynamic assimilation, were proposed. The multi-paradigm platform was validated by conducting melanoma chemotaxis under foetal bovine serum gradient. Due to its CFD and agent-based modalities, the platform can be employed as both a design optimisation as well as hypothesis testing tool.

Orientador: Ibsen Bellini Coimbra<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas<br>Made available in DSpace on 2018-08-21T06:48:06Z (GMT). No. of bitstreams: 1 Bombini_MarianaFreschi_M.pdf: 1816531 bytes, checksum: 7a3edba503588d6825d034f24c884991 (MD5) Previous issue date: 2012<br>Resumo: A utilização de células-tronco mesenquimais (CTM) para a reconstrução da cartilagem articular é uma promissora alternativa terapêutica, devido à vulnerabilidade do tecido a lesões e processo degenerativo irreversível. O objetivo deste estudo foi investigar o potencial condrogênico de CTM de líquido amniótico humano (CTM-LA) em sistema de cultura de micromass (alta densidade celular) com TGF-?3 por 21 dias. Métodos: 53 líquido amnióticos (LA) foram obtidos de mulheres submetidas à amniocentese durante o segundo trimestre de gravidez. A indicação da amniocentese foi feita pela obstetrícia, conforme protocolo específico do serviço de medicina fetal da UNICAMP. Foram selecionadas células-tronco mesenquimais, caracterizadas por citometria de fluxo. Estas células foram expandidas para obter um número populacional para o desenvolvimento do micromass. O micromass foi realizado em placa de cultura de 96 poços com fundo em "v", em cada poço foram pipetados 10?l contendo 5x105 CTM-LA e meio para diferenciação condrogênica contendo TGF-?3. Esta condição se manteve por 21 dias, e então, o potencial condrogênico foi avaliado pela presença da proteína do colágeno II pela técnica de western blotting, também foi avaliada a expressão gênica do Sox-9, colágeno II e agrecano pela técnica da PCR em tempo real (qRT-PCR). Comparamos CTM-LA em monocamada a CTM-LA submetidas ao sistema de cultura de micromass e como controle positivo utilizamos a cartilagem adulta humana. Resultados: Confirmamos o potencial condrogênico pela diferenciação das CTM-LA em condrócitos através da expressão dos genes SOX-9, colágeno tipo II e agrecano, bem como a proteína do colágeno II. A expressão de SOX-9 em micromass foi significativamente maior do que na cartilagem adulta. Conclusão: A condrogênese foi desenvolvida a partir da combinação de uma fonte de célula tronco recém descrita proveniente do líquido amniótico humano com o sistema de cultura de micromass. Esta fonte apresentou alto potencial condrogênico e dessa forma, fortes evidências para aplicações clínicas. Os resultados são promissores e sugerem a possibilidade de investimentos em bancos de líquido amniótico<br>Abstract: Introduction: The use of mesenchymal stem cells (MSC) for reconstruction of articular cartilage, leads to a promising therapeutic alternative, due to the tissue vulnerability to injuries and irreversible degenerative process. The aim of this study was to investigate the chondrogenic potential of MSC from human amniotic fluid in Micromass system (high-density cell culture) with TGF-?3 for 21 days. Methodology: The amniotic fluid was obtained from 53 pregnant women. The micromass was performed using MSC that was cultured in monolayer and chondrocytes from adult human normal cartilage as control groups. After 21 days, the chondrogenic potential was determined by metabolic products released from the cell, such as SOX-9, type II collagen and aggrecan. This study was approved by the ethics committee. Results: The proteic production of type II collagen was observed by Western Blotting. The genetic expression of SOX-9 was analyzed by PCR in real time, and this was found to be significantly higher than in adult cartilage. The same procedure was used to determinate the genetic expression of aggrecan and type II collagen, verifying positive result for both. Conclusion: Chondrogenesis was developed from the unique combination of the newly discovered source of mesenchymal stem cells from human amniotic fluid with micromass, and it demonstrated a satisfactory expression. Thus, this source is extremely viable for clinical applications, and the results suggest the possibility of investments in human amniotic fluid banks<br>Mestrado<br>Clinica Medica<br>Mestra em Clínica Médica

INTRODUÇÃO: O fenótipo de resistência a drogas é caracterizado pela superexpressão das proteínas da família ABC (ATP-Binding Cassette). A Pglicoproteína (P-gp), codificada pelo gene ABCB1, é uma das bombas de efluxo mais estudadas, como agente interferente no tratamento de diversos tipos de câncer, seguida pela proteína MRP1 (Multidrug Resistance-related Protein 1), transcrita pelo gene ABCC1. Estudos recentes relacionaram a atuação da proteína LRP (Lung Resistance Protein) a estas bombas, devido sua alta expressão em tumores com fenótipo resistente. Em contrapartida, estes transportadores também exercem funções fisiológicas contra metabólitos, compostos citotóxicos e teratogênicos, em diversos tecidos normais como rins, fígado, intestino e célulastronco. As proteínas ABC são consideradas marcadores específicos das célulastronco hematopoiéticas, porém, ainda são pouco descritas nas células-tronco mesenquimais (CTM). A medula óssea (MO) é a fonte mais bem descrita de CTM adultas, cujas propriedades são conhecidas e utilizadas na aplicação clínica. Entretanto, recentemente células-tronco de origem fetal têm criado expectativas e o líquido amniótico (LA) apontado como fonte promissora deste tipo celular, que por sua vez, é pouco estudada acerca da atuação das bombas ABC. Sendo assim, o presente estudo visou caracterizar a expressão da P-gp, MRP1 e LRP em célulastronco mesenquimais humanas do líquido amniótico e medula óssea. MÉTODOS: Foram isoladas CTM de amostras do LA e MO, caracterizadas através de citometria de fluxo, ensaios de diferenciação em tecidos mesenquimais in vitro e expressão dos genes de indiferenciação Oct-4 e Nanog por RT-PCR. Verificou-se também a presença da P-gp através da técnica de imunocitoquímica e a sua resistência frente a diferentes concentrações de doxorrubicina (DOX) através do ensaio de MTT, foram utilizadas como controles as células MES-SA e MES-DX (sarcoma uterino respectivamente sensível e sua variante resistente à DOX). Para avaliar o funcionamento da P-gp, foi feito ensaio de exclusão do corante Rhodamina 123 (Rh 123) por meio de citometria de fluxo, com ou sem bloqueio da P-gp utilizando verapamil. E por fim, foi verificada a expressão dos genes ABCB1/ABCC1/LRP por meio de PCR em tempo real, nas amostras pré e pós-diferenciações adipogênica, osteogênica e condrogênica. RESULTADOS: As CTM, tanto da MO quanto do LA, exibiram respostas semelhantes às células resistentes MES-DX e expressam a Pgp de forma homogênea nas suas populações. O ensaio de exclusão da Rh 123 demonstrou dinâmicas de efluxo do corante semelhantes entre as células-tronco do LA e as MES-DX, com e sem a presença de verapamil. No entanto, as células da MO apresentaram efluxo crescente e não responderam ao bloqueador verapamil como as outras linhagens. A distribuição da expressão gênica, o ABCB1 se mostrou menor que a do LRP nas amostras de LA indiferenciadas. No entanto a expressão do ABCB1 nas amostras de LA foi maior em comparação às amostras de MO indiferenciadas. Não houve seignificância estatística na comparação da expressão gênica antes e depois das diferenciações adipogênica e osteogênica. CONCLUSÃO: As CTM são resistentes ao quimioterápico doxorrubicina, mas, possuem baixa expressão do ABCB1. Portanto, as CTM podem possuir outro mecanismo, como a MRP1 e LRP, atuando no mecanismo de resistência à DOX, além da P-gp<br>BACKGROUND: The drug resistance phenotype is characterized by ABC (ATPBinding Cassette) family proteins overexpression. The P-glycoprotein (P-gp) codified by ABCB1 gene is one of the most studied efflux pumps such as interfering agent in cancer treatment followed by MRP1 (Multidrug Resistance-related protein 1) transcribed by ABCC1 gene. Recent studies have linked the LRP (Lung Resistance Protein) to these pumps activities because of its high expression in resistant cancers. Though these transporters also have physiological functions against metabolites, cytotoxic and teratogenic compounds in normal tissues as kidneys, liver, intestine and stem cells. ABC proteins are considered hematopoietic stem cells specific markers but are poorly described in mesenchymal stem cells (MSC). Bone marrow (BM) is the best characterized adult MSC source its properties are well known and used in clinical application. However recently fetal stem cells has raised expectations and amniotic fluid (AF) is a promising source of this cell type which in turn is scarce regarding about presence and activity of the ABC pumps. The aim of this study was characterize the P-gp, MRP1 and LRP expression in human mesenchymal stem cells from amniotic fluid and bone marrow. METHODS: Mesenchymal stem cells were isolated from AF and BM and characterized by flow cytometry, in vitro mesenchymal differentiation assays, Oct-4 and Nanog genes expression by RT-PCR. The P-gp presence were found over immunocytochemical technique and its activity against different concentrations of doxorubicin (DOX) by MTT assay which were used as control cells MES-DX and MES-SA (uterine sarcoma respectively resistant and susceptible to DOX). The P-gp was evaluated in Rhodamine 123 (Rh 123) dye exclusion through flow cytometry with or without blocking P-gp from verapamil. Finally was observed ABCB1/ABCC1/LRP genes expression by real time PCR after and before adipogenic, osteogenic and chondrogenic differentiation. RESULTS: BM and AF MSC showed the same response of the DOX resistant cells MES-DX and express P-gp homogeneously though their populations. The Rh 123 dye exclusion assay demonstrated that the AF stem cells efflux dynamics are similar to the MES-DX with and without the presence of verapamil. However BM MSC showed crescent efflux and no response to verapamil as the other cells. The ABCB1 gene was less expressed than LRP in undifferentiated AF MSC. No difference was found in gene expression before osteogenic and adipogenic differentiation. CONCLUSION: MSC have low expression of ABCB1 although are resistant to doxorubicin so other mechanisms such as MRP1 or LRP may be acting to these cells defense in addition to P-gp

Le cartilage articulaire est un tissu avasculaire et non innervé, ce qui lui confère des capacités de réparation très limitées. Les traitements chirurgicaux actuels ne permettent pas d’obtenir un tissu de réparation similaire au cartilage natif. Les recherches s’orientent depuis de nombreuses années vers l’ingénierie cellulaire et tissulaire du cartilage selon le type de lésions à traiter, focale ou diffuse. Les cellules souches mésenchymateuses (CSMs) constituent une source cellulaire intéressante pour l’ingénierie du cartilage. Elles sont facilement accessibles et ont des potentialités de différenciation chondrogénique. Les CSMs issues de la moelle osseuse sont les plus étudiées et constituent la référence. D’autres sources de CSMs sont également très prometteuses. Notre choix s’est porté sur les CSMs issues de la membrane et du liquide synovial. Ces deux tissus articulaires présentent l’avantage de pouvoir être prélevés facilement lors d’un examen arthroscopique et leurs CSMs sont adaptées au microenvironnement (hypoxie, inflammation) de l’articulation. Ce travail a porté sur l’étude de deux sources cellulaires d’origine synoviale dans le traitement des lésions focales et diffuses du cartilage. Ces CSMs d’origine synoviale ont d’abord été caractérisées selon leurs phénotypes et leurs capacités de différenciation vers les voies ostéogénique, adipogénique et chondrogénique, par rapport aux CSMs issues de la moelle osseuse. Ensuite, les capacités chondrogéniques de ces CSMs synoviales destinées à produire un substitut cartilagineux consacré aux traitements des lésions focales du cartilage articulaire ont été étudiées. Les CSMs ont été ensemencées dans un biomatériau collagénique et différentes conditions environnementales (facteurs de croissance et oxymétrie) ont été évaluées afin de définir les conditions de culture les plus appropriées. La chondrogenèse a été induite avec succès par l’utilisation de facteurs de croissance tels que TGF-1 et TGF-3 seuls ou en association avec la BMP-2. L’hypoxie n’a pas montré d’effet bénéfique sur la synthèse matricielle au sein des substituts cartilagineux. Enfin, nous avons évalué les capacités des CSMs issues du liquide synovial à traiter les lésions diffuses du cartilage induites par un modèle de section du ligament croisé antérieur chez le rat athymique. Les deux injections intra-articulaires de CSMs issues du liquide synovial à 1 et 2 semaines après chirurgie n’ont pas permis de prévenir les lésions arthrosiques<br>Joint cartilage is avascular and not innervated, which gives it very limited repair capabilities. Current surgical treatments do not provide repair tissue similar to native cartilage. For many years, research has been focused on cellular and tissue engineering of cartilage depending on the type of lesions to be treated, focal or diffuse. Mesenchymal stem cells (MSCs) are an interesting cellular source for cartilage engineering. They are easily accessible and have the potential for chondrogenic differentiation. MSCs from bone marrow are the most studied and are the gold-standard. Other MSCs sources of are also very promising. We chose MSCs from the synovial membrane and synovial fluid. These both joint tissues have the advantage of being easily retrievable during arthroscopic examination and their MSCs are adapted to the microenvironment (hypoxia, inflammation) of the joint. This thesis work focused on the study of two cellular sources of synovial origin in the treatment of focal and diffuse cartilage lesions. These synovial-derived MSCs were first characterized according to their phenotypes and their ability to differentiate to the osteogenic, adipogenic and chondrogenic pathways, compared to bone marrow derived MSCs. Then, the chondrogenic capacities of these synovial MSCs to produce a cartilage substitute for the treatment of focal lesions of joint cartilage were studied. The MSCs were seeded in a collagenic biomaterial and different environmental conditions (growth factors and oximetry) were evaluated to define the most appropriate culture conditions. Chondrogenesis has been induced with success by the use of growth factors such as TGF-β1 or TGF-β3 alone or in combination with BMP-2. Hypoxia has not exerted a beneficial effect on matrix synthesis in cartilage substitutes.Finally, we evaluated the ability of CSMs from human synovial fluid to treat diffuse cartilage lesions induced by an anterior cruciate ligament section model in athymic rats. The two intra-articular injections of synovial fluid MSCs, 1 and 2 weeks after surgery did not prevent osteoarthritic lesions

La polyarthrite rhumatoïde (PR) est une maladie auto-immune inflammatoire caractérisée par une synovite chronique aboutissant à la dégradation du cartilage et une érosion osseuse. Malgré la présence de stratégies thérapeutiques capables de contrôler la maladie, il n'existe aujourd'hui aucun traitement curatif. De nombreuses études ont déjà démontré les capacités immunomodulatrices des cellules souches mésenchymateuses dérivées du tissu adipeux (ADSC) in vitro et dans des modèles murins de PR. Cependant, cet effet n'a toujours pas été traduit efficacement dans un contexte clinique. L'objectif de cette étude est de définir comment l'inflammation pourrait dicter l'efficacité thérapeutique des ADSC par (i) une approche translationnelle en utilisant des liquides synoviaux (SF) de patients atteints de PR ou (ii) en étudiant l'effet des injections d'ADSC dans deux modèles différents d'arthrite expérimentale. Nous montrons que les SF pro-inflammatoires conservent la capacité des ADSC à proliférer ainsi que leur phénotype et induisent l'expression des gènes COX-2, IDO, IL-6, TSG6, ICAM-1, VCAM-1 and PD-L1 qui sont tous impliqués dans le potentiel immunomodulateur des ADSC. Nous démontrons que l'effet des SF est directement médié par la voie de signalisation TNF/NF-?B et partiellement par l'IL-6. De plus, le pré-traitement des ADSC avec les SF inflammatoires renforce leur capacité à moduler les marqueurs pro-inflammatoires des macrophages (CD40 et CD80) et à induire des lymphocytes T régulateurs (Tregs). In vivo, les ADSC injectées par voie intra-péritonéale ont aggravé la progression d'arthrite dans le modèle de transfert de sérum K/BxN en traitement préventif mais n'ont eu aucun effet sur les scores arthritiques en traitement thérapeutique. En outre, un effet protectif sur la progression de l'arthrite n'a été observé que lorsque les ADSC ont été injectées par voie intra-articulaire. L'ensemble de nos résultats mettent en avant l'influence de l'environnement inflammatoire local sur les effets thérapeutiques des ADSC et suggèrent leur adressage direct aux sites inflammatoires qui stimulent leur potentiel immunomodulateur et maximisent leur intérêt clinique<br>Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by chronic synovitis that leads to bone erosion and cartilage degeneration. Despite the presence of therapeutic strategies capable of managing disease progression, there is currently no cure available. Adipose-derived mesenchymal stem cells (ADSC) have been successfully shown to have immunomodulatory abilities both in vitro and in animal models of RA. However, this effect has yet to be efficiently translated in clinical settings. In the present study, we aimed to define how inflammation could play part in dictating the therapeutic efficiency of ADSC through either (i) a translational approach by using synovial fluids from RA patients (RASF) or (ii) by studying the effect of ADSC injections in two different RA experimental models. Here, we show that pro-inflammatory RASF preserve ADSCs' proliferative capacity and phenotype and up-regulates the gene expression of COX-2, IDO, IL-6, TSG6, ICAM-1, VCAM-1 and PD-L1 which are all involved in the immunomodulatory potential of ADSC. We demonstrate that RASF effect is directly mediated by TNF/ NF-?B signaling pathway and partially by IL-6. Moreover, pre-treating ADSC with inflammatory RASF functionally enhances their ability to alter pro-inflammatory markers in macrophages (CD40 and CD80) and induce regulatory T cells (Tregs). In vivo, ADSC worsened disease progression in the K/BxN serum transfer arthritis model when injected intraperitoneally as a preventive protocol but did not have any effect on arthritic scores when injected as a therapeutic protocol. Only when ADSC were injected intra-articularly did we observe protective effects on the progression of arthritis. Altogether, this work puts forward the influence of the local inflammatory environment on ADSC therapeutic effects and suggests a direct delivery to sufficiently inflamed sites in order to properly prime their immunomodulatory potential and maximize their clinical benefits

La transplantation rénale, thérapie de choix de l'insuffisance rénale chronique terminale, est limitée par une pénurie d'organes. Les greffons issus de donneurs décédés par arrêt cardiaque (DDAC) peuvent contribuer à pallier à cette pénurie au prix de stratégies thérapeutiques visant à améliorer l'issue de la transplantation. Les cellules souches mésenchymateuses (MSC) de l'organisme adulte ont des propriétés de sécrétion, d'immunomodulation et de différenciation intéressantes dans ce contexte.L'objectif de ce travail est d'évaluer, dans un modèle pré-clinique porcin de DDAC, le potentiel thérapeutique de deux populations cellulaires d'intérêt : les MSC issues du liquide amniotique (AFSC) et les cellules de la fraction vasculaire stromale du tissu adipeux (SVF). Les AFSC porcines injectées dans l'artère rénale 7 jours post-greffe, en raison de leur sensibilité à une séquence d'hypoxie réoxygénation (HR) in vitro, accélèrent la reprise de fonction et réduisent l'extension des lésions chroniques du greffon et sont détectées dans le rein 24h après injection. La SVF porcine, phénotypiquement proche de celle de l'Homme, est moins sensible à cette séquence d'HR et peut être injectée dans l'artère du greffon à sa reperfusion sans perturbation du flux sanguin rénal, avec une rétention des cellules dans le rein 24h post injection.Ce travail met en évidence le rôle bénéfique des AFSC dans la réparation des lésions ischémie-reperfusion des greffons issus des DDAC, ainsi que la faisabilité de l'injection de la SVF dans l'artère rénale après transplantation, et ouvre des pistes pour l'optimisation les protocoles d'administration de produits de thérapie cellulaire en transplantation<br>Kidney transplantation is the best therapeutic option for end stage chronic kidney failure, but is limited by transplant shortage. Use of transplants from deceased after cardiac death donors (DCD) could represent an additional graft source, but there is a need for developing new therapeutic approaches like cell therapy to increase their recovery. Mesenchymal stem cells (MSC) potentially extracted from many adult tissues have interesting paracrine, immune-modulating, and differentiation properties in this context. This work aims to assess, in a preclinical porcine model DCD donor, the therapeutic potential of two cell populations of interest: amniotic fluid derived MSC (AFSC) and cells from stromal vascular fraction of adipose tissue (SVF). Delayed injection of AFSC 7 days following kidney transplantation because of their sensitivity to a specific Hypoxia Reoxygenation (HR) sequence in vitro, accelerates graft function recovery and limits chronic injuries to the transplanted organ. Cells are detectable into the transplanted kidney 24h after injection. Porcine SVF is phenotypically similar to human. Injected in renal artery simultaneously with organ reperfusion because of its resistance to the HR sequence, porcine SVF does not disturb renal blood flow and allow cell-retention within the organ 24h after injection. This work highlights the protective effect of AFSC against ischemia reperfusion lesions in grafts from DCD donors and the feasibility of SVF injection directly into the renal artery of the graft following kidney transplantation in DCD conditions. Moreover it opens new lines for optimizing injection protocols of cellular products in kidney transplantation

博士<br>國立成功大學<br>臨床醫學研究所<br>101<br>Artery occlusive diseases, including coronary artery disease, peripheral artery occlusive disease, and cerebrovascular disease, are major health issues that affect a large proportion of the adults, especially among aging population. Although there are several therapeutic strategies for artery occlusive diseases, i.e. optimal medical treatment and revascularization either by endovascular intervention or by open surgery, the prognosis is not satisfied. Therefore, how to improve blood perfusion in ischemic tissues should play a critical role in improving prognosis of patients with artery occlusive diseases. Previous studies highlighted the necessity of revascularization and reported that bone marrow-derived progenitor cells significantly enhanced cardiac repair by promoting neoangiogenesis. Consequently, to achieve efficient revascularization in ischemic regions is essential for restoring blood perfusion and physiologic function. Angiogenesis is a tightly controlled process where endothelial cells proliferation and migration is regulated by secreted factors as well as by surrounding cells and matrix. However, mature endothelial cells possess limited regenerative capacity. Recently, mounting evidence showed that stem cells, such as endothelial progenitor cells, bone marrow stem cells, or cord blood stem cells, has been used to induce neovascularization in animal models of limb and myocardial ischemia, but unfortunately, the therapeutic efficacy of these stem cells is controversial. It is well-known that human embryonic stem cell (hESC) is pluripotent and can differentiate into a wide range of cell types representing the 3 primary embryonic lineages of mesoderm, ectoderm, and endoderm. However, the use of hESCs for research or therapeutic purposes has been constrained by several issues: complex social and ethical considerations, immune rejection reactions, and tumors formation in recipients. Amniotic fluid-derived stem cells (AFSCs) are known to have a high renewal capacity and great differentiation potency. Unlike hESCs, the AFSCs do not form tumors in severe combined immunodeficient mice, and ASFC research does not raise profound ethical issues. Atala and his colleagues demonstrated that AFSCs are pluripotent and can be directed into a wide spectrum of cell types. Therefore, we conducted this study to test whether AFSCs can function as a cell source for therapeutic angiogenesis in a mouse hindlimb ischemia model: (1) to prove in vitro that AFS cells can be differentiated into endothelial lineage cell (EC), (2) to examine the in vivo therapeutic efficacy of AFSC or AFSC- derived EC (AFSC-EC) transplantation in a nude mice model of hindlimb ischemia, and (3) to identify the possible mechanisms of neoangiogenesis induced by AFSCs or AFSC-ECs. We differentiated human AFSCs into ECs in vitro, as evidenced by expression of endothelial cell markers, and capillary-like network formation on Matrigel. One day after high ligation of the external iliac artery in athymic nude mice, AFSC-ECs were intramuscularly injected into ischemic limbs. As compared to AFSCs, HUVECs, and medium, intramuscular AFSC-EC injection into the ischemic regions improved limb salvage, restored blood perfusion, and significantly increased capillary and arteriole densities. We did not find any evidence of acute rejection or tumor formation in nude mice after intramuscular AFSC-EC injection, and most importantly, transplanted AFSC-ECs survived in the ischemic tissue. Those transplanted AFSC-ECs were incorporated into vessels in the ischemic region, as confirmed by immunofluorescent staining for human smooth muscle 22α or von Willebrand factor. Matrix metalloproteinase (MMP)-9 might stimulate VEGF-A release from AFSC-ECs and thus induce neovascularisation. Our study indicates that AFSC-ECs were a suitable cell source for the treatment of artery occlusive diseases with therapeutic angiogenesis. AFSC-EC transplantation has the potential to promote therapeutic angiogenesis in vivo by facilitating neovascularization in a mouse model of ischemia. We conclude that AFSC-ECs can act as a novel source of xenograft, even allograft, transplantation cells for therapeutic angiogenesis in the treatment of ischemic diseases. In the future, AFSCs might be useful for autologous as well as nonautologous regenerative therapies via matching of histocompactible donor cells with recipients.

The amniotic membrane and the amniotic fluid are one of life's most complex and delicate tissues and fluids, respectively. What was known about this tissue and fluid prior to the 20th century was extremely limited scientifically, but was significantly defined by beliefs entrenched in mysticism, folklore, and superstitions. A comprehensive literature review of the amniotic membrane tissue and amniotic fluid reveals the many unique and complex characteristics and biological properties that been heavily investigated since the turn of the 20th century and continues to surge into the 21st century. The historical perspectives, evolution, derivation, histology, structure, and composition of the amniotic membrane; and historical perspectives, volume and regulation, and cellular and non-cellular composition of the amniotic fluid are discussed here and are coalesced for an easy and comprehensible resource. Lastly, future perspectives regarding research and application of the amniotic membrane and amniotic fluid, including stem cells are discussed.

碩士<br>國立臺灣大學<br>應用力學研究所<br>96<br>Research of stem cells for cardiovascular diseases is of growing interest. The miniaturized and portable micro bio-devices fabricated by micromachining technology is able to provide in-depth understanding of biological mechanism and activities related to cardiovascular diseases. The objective of this research is to study the influence of microfluidic shear stress to the culture of ECs, and AFSC differentiation to ECs. Design and fabrication of micro devices are key to success for the experiment, as well as the integration of real-time monitoring optical and culturing systems. The experiment of cell culture was carried out over 24 hours. It was found that, under the microfluidic stimulation of shear stress, endothelial cells were successfully adhered on the substrate, stretched and aligned along with constant flows applied. The microfluidic shear stresses were made at 3, 6, 12, 18 dynes/cm2, respectively. This work shows the result of cell morphology and cell arrangement under the fluidic shear stress. 80% of endothelial cells were mostly found in morphology and arrangement aligned along the flow direction. Meanwhile, stretched cells were found in less than 20° out of the flow direction at the fluidic shear stress of 12 ynes/cm2. In addition, the migration speed of endothelial cells was also measured to be about 0.4 um/min. at the shear stress of 3 and 6 dynes/cm2, and 0.8 um/min. at the shear stress of 12 and 18 dynes/cm2, respectively. Biological and fluidic stimulations of AFSC ifferentiation to endothelial cells were also conducted. vWF bio markers were first found in micro devices to provide initial evidence of AFSC differentiation to endothelial cells. At last, the device and system were successfully established and utilized for its potential of AFSC differentiation to endothelial cells for cardiovascular diseases.