Thèses sur le sujet « Cellule Fetali »

La ricerca sulle cellule staminali apre nuove prospettive per approcci di terapia cellulare. Molta attenzione è concentrata sulle cellule staminali isolate da membrane fetali, per la facilità di recupero del materiale di partenza, le limitate implicazioni etiche e le caratteristiche delle popolazioni di cellule staminali residenti. In particolare a livello dell’epitelio amniotico si concentra una popolazione di cellule (hAECs) con interessanti caratteristiche di staminalità, pluripotenza e immunomodulazione. Restano però una serie di limiti prima di arrivare ad un’applicazione clinica: l’uso di siero di origine animale nei terreni di coltura e le limitate conoscenze legate alla reazione immunitaria in vivo. La prima parte di questo lavoro è focalizzata sulle caratteristiche delle hAECs coltivate in un terreno privo di siero, in confronto a un terreno di coltura classico. Lo studio è concentrato sull’analisi delle caratteristiche biologiche, immunomodulatorie e differenziative delle hAECs. L’interesse verso le caratteristiche immunomodulatorie è legato alla possibilità che l’uso di un terreno serum free riduca il rischio di rigetto dopo trapianto in vivo. La maggior parte degli studi in vivo con cellule isolate da membrane fetali sono stati realizzati con cellule di derivazione umana in trapianti xenogenici, ma poco si sa circa la sopravvivenza di queste cellule in trapianti allogenici, come nel caso di trapianti di cellule di derivazione murina in modelli di topo. La seconda parte dello studio è focalizzata sulla caratterizzazione delle cellule derivate da membrane fetali di topo (mFMSC). Le caratteristiche biologiche, differenziative e immunomodulatorie in vitro e in vivo delle mFMSC sono state confrontate con i fibroblasti embrionali di topo. In particolare è stata analizzata la risposta immunitaria a trapianti di mFMSC nel sistema nervoso centrale (CNS) in modelli murini immunocompetenti.
The stem cell research opens new perspectives for cell therapy approaches. Much attention has been focused on stem cells isolated from fetal membranes, for the easy recovery of these tissues, the limited ethical implications and the characteristics of resident stem cells. In particular from the amniotic epithelium it is possible to isolate a population of cells (hAECs) with interesting characteristics of stemness, pluripotency and immunomodulation. However, before going to clinic there are some limitations to overcome: the use of culture media supplemented with animal serum and the limited knowledge related to immune reactions in vivo. The first part of this work is focused on the characterization of hAECs cultured in a serum-free medium, in comparison to a classical culture medium. The study is concerned with the biological, immunomodulatory and differentiation properties of hAECs. The interest towards immunomodulatory characteristics is related to the possibility that using a serum free medium could reduce the risk of grafts rejection after transplantation in vivo. The majority of in vivo studies with cells isolated from fetal membranes were carried out with human-derived cells in xenogeneic transplantation, but little is known about the survival of these cells in allogeneic settings, as for transplantation of murine cells in mouse models. The second part of this study is focused on the characterization of cells derived from murine fetal membranes (mFMSC). Biological characteristics, differentiation potential, in vitro and in vivo immunomodulatory properties of mFMSC has been compared with mouse embryonic fibroblasts. In particular we analyzed the immune response towards mFMSC grafted in the central nervous system (CNS) of immunocompetent mice.

La ricerca sulle cellule staminali apre nuove prospettive per approcci di terapia cellulare. Molta attenzione è concentrata sulle cellule staminali isolate da membrane fetali, per la facilità di recupero del materiale di partenza, le limitate implicazioni etiche e le caratteristiche delle popolazioni di cellule staminali residenti. In particolare a livello dell’epitelio amniotico si concentra una popolazione di cellule (hAECs) con interessanti caratteristiche di staminalità, pluripotenza e immunomodulazione. Restano però una serie di limiti prima di arrivare ad un’applicazione clinica: l’uso di siero di origine animale nei terreni di coltura e le limitate conoscenze legate alla reazione immunitaria in vivo. La prima parte di questo lavoro è focalizzata sulle caratteristiche delle hAECs coltivate in un terreno privo di siero, in confronto a un terreno di coltura classico. Lo studio è concentrato sull’analisi delle caratteristiche biologiche, immunomodulatorie e differenziative delle hAECs. L’interesse verso le caratteristiche immunomodulatorie è legato alla possibilità che l’uso di un terreno serum free riduca il rischio di rigetto dopo trapianto in vivo. La maggior parte degli studi in vivo con cellule isolate da membrane fetali sono stati realizzati con cellule di derivazione umana in trapianti xenogenici, ma poco si sa circa la sopravvivenza di queste cellule in trapianti allogenici, come nel caso di trapianti di cellule di derivazione murina in modelli di topo. La seconda parte dello studio è focalizzata sulla caratterizzazione delle cellule derivate da membrane fetali di topo (mFMSC). Le caratteristiche biologiche, differenziative e immunomodulatorie in vitro e in vivo delle mFMSC sono state confrontate con i fibroblasti embrionali di topo. In particolare è stata analizzata la risposta immunitaria a trapianti di mFMSC nel sistema nervoso centrale (CNS) in modelli murini immunocompetenti.
The stem cell research opens new perspectives for cell therapy approaches. Much attention has been focused on stem cells isolated from fetal membranes, for the easy recovery of these tissues, the limited ethical implications and the characteristics of resident stem cells. In particular from the amniotic epithelium it is possible to isolate a population of cells (hAECs) with interesting characteristics of stemness, pluripotency and immunomodulation. However, before going to clinic there are some limitations to overcome: the use of culture media supplemented with animal serum and the limited knowledge related to immune reactions in vivo. The first part of this work is focused on the characterization of hAECs cultured in a serum-free medium, in comparison to a classical culture medium. The study is concerned with the biological, immunomodulatory and differentiation properties of hAECs. The interest towards immunomodulatory characteristics is related to the possibility that using a serum free medium could reduce the risk of grafts rejection after transplantation in vivo. The majority of in vivo studies with cells isolated from fetal membranes were carried out with human-derived cells in xenogeneic transplantation, but little is known about the survival of these cells in allogeneic settings, as for transplantation of murine cells in mouse models. The second part of this study is focused on the characterization of cells derived from murine fetal membranes (mFMSC). Biological characteristics, differentiation potential, in vitro and in vivo immunomodulatory properties of mFMSC has been compared with mouse embryonic fibroblasts. In particular we analyzed the immune response towards mFMSC grafted in the central nervous system (CNS) of immunocompetent mice.

Cell therapy is an attractive perspective for the treatment of life threatening disorders. In this context, foetal tissues are gaining interest as sources of cells for auto- and allo-transplantation, because of their pluripotency, proliferative capability and their low, if any, immunogenicity. Recently a pluripotent stem cell population has been isolated from Amniotic Fluid (AF). It is able to proliferate for more than 18 months, maintaining their differentiative ability as well as a normal karyotype. In term of differentiation potential, we succeeded in obtaining in vitro mesenchymal-, ectodermal- and endodermal-derived tissues from human Amniotic Fluid Stem (AFS) cells. Furthermore, murine AFS cells injected in blastocytes took part to the formation not only of several different foetal organs, but also of the placenta and the umbilical cord. In the present study we investigated the possibility of differentiating AFS cells towards the hematopoietic pathway. AFS cells isolated from human amniotic fluid, collected during routine diagnostic procedures and obtained under informed consent, were firstly expanded in vitro and selected on the basis of their ckit expression. We achieved a reproducible erythroid differentiation by culturing hAFSCs as embryoid bodies (EBs) under serum free conditions with haematopoietic cytokines. Erythroid cells expressing CD235a constituted 70% of the total hAFSCs forming EBs showing also a co-expression of CD36 and CD71. Furthermore, human erythrocytes (human CD235a) were isolated from bone marrow and spleen of sublethally irradiated NOD/SCID mice at 3 months after the injection of hAFSCs. To determine if the expansion procedure had led to a restriction of the hematopoietic potential towards the erythroid pathway, we compared expanded AFSCs and freshly isolated cKit+ Lin- (AFKL) cells. We also harvested cKit+ Lin- KL cells from the membrane surrounding the AF, the Amnion, in search for a possible origin. We compared the hematopoietic potential of mAFKL and mAmKL to Fetal Liver KL, the main source of fetal HSC. When cultivated immediatly after their sorting, freshly isolated murine AFKL and AmKL cells gave rise to all the different hematopoietic lineages both in vitro and in vivo. Actually, when cocultivated with OP9(d)1 cells, AFKL and AmKL undergo complete T cell differentiation within 2 weeks. They also generate myeloid and erythroid colonies when cultivated in methylcellulose for clonogenic assay. The erythroid restricted potential of human AFS cells was thus probably linked to the in vitro expansion procedure. Moreover, cells belonging to all the three hematopoietic lineages (lymphoid, myeloid and erythroid) and arising from freshly isolated mAFKL and mAmKL are found in the peripheral blood of sublethally irradiated RAG1 deficient mice only 4 weeks after transplantation. Four month later, transplanted mice showed mAFKL-derived lymphoid, myeloid and erythroid cells, in all the hematopoietic organs. Successful econdary transplantation strongly suggest that mAFKL and mAmKL comprise HSC, with self-renewal ability. Those results were very similar to those obtained with mFLKL, confirming the strong hematopoietic potential of mAFKL and mAmKL. Experiments with freshly isolated hAFKL gave good results in the in vitro assays being able to give rise to erythroid, myeloid and lymphoid lineages, but failed to reconstitute the hematopoietic system in irradiated NOD/SCID mice, probably due to the poor amount of cells injected. This is the first report demonstrating that AFKL and AmKL do have an haematopoietic potential, supporting the idea that AF and Am may be an excellent source for therapeutic application.

The work here presented is a part of a bigger project at the Centro di Ricerca E. Menni (Fondazione Poliambulanza, Istituto Ospedaliero, Brescia), aimed to identify new adult stem cells sources. In these last years, in order to find a tissue easy to procure and that allows to obtain high cell yeld researchers have turned their attention to alternative sources of adult stem cells as adipose tissue, peripheral blood, cord blood and, more recently, placenta tissue. In this field, placenta is of particular interest either because of its immunological role in maintaining fetus-maternal tolerance either because of its early embryological origin which may entail an immature phenotype. In particular we are focusing our attention on fetal membranes, amnion and chorion, which form the outer limit of the sac that encloses the fetus. The amnion is an unique avascular tissue and is comprised of an epithelial monolayer on the top of a mesodermal layer. The amniotic mesoderm is loosely connected to the chorionic mesoderm, in contact to the trophoblastic layer which is in intimate contact with the maternal decidua. The aim of this study was to isolate different cell populations from the 4 layers which costitute the fetal membranes, evaluating all the features that contribute to hypothesize the presence of stem cells. In particular the phenotypic profile and the differentiation potential toward mesodermal lineages of the different cell populations were evaluated, in comparison with bone marrow stromal cells.

Attualmente i metodi di diagnosi prenatale esistenti, consentono di ottenere cellule fetali solamente mediante metodiche invasive che comportano un rischio sia per la madre che per il feto. La scoperta nel 1979 della presenza di cellule fetali circolanti nel sangue materno, ha aperto la strada per lo sviluppo di metodi non invasivi per l’identificazione di anomalie fetali. Tutti i metodi sviluppati fino ad oggi tuttavia sono risultati inefficienti nel garantire un adeguato numero di cellule fetali, essendo estremamente laboriosi, richiedendo molto tempo ed essendo operatori-dipendenti. Questo progetto ha lo scopo di sviluppare un metodo non invasivo per l’isolamento di singole cellule fetali dal sangue materno, per una analisi diretta dei cromosomi fetali. La prima parte è stata dedicata alla ricerca e test di diversi marcatori specifici per l’arricchimento e l’identificazione delle cellule fetali. Una volta messo a punto lo step di arricchimento, quest’ultimo è stato implementato e integrato in un worklow più ampio che prevedeva: prelievi di sangue da donne in gravidanza, arricchimento positivo magnetico, marcatura delle cellule fetali, isolamento come singole cellule e analisi genetica. Appena l’intero flusso è stato standardizzato abbiamo cominciato una valutazione clinica su donne in gravidanza. Al fine di determinare la percentuale di successo e il numero di cellule fetali per campione, un totale di 372 donne sono state reclutate e stratificate in base alla loro settimana gestazionale al momento del prelievo. Al meno una cellula fetale è stata isolata nel 90.7% dei casi prelevati tra la decima e undicesima settimana gestazionale, con un numero medio totale di 3.5 cellule fetali per paziente. Inoltre dati preliminari ottenuti da 131 donne che venivano sottoposte all’esame invasivo, hanno mostrato un alto livello di concordanza tra le cellule isolate singolarmente e analizzate con la nostra metodica e i risultati derivanti dall’esame diagnostico. Complessivamente, i risultati derivanti da questo studio, supportano la fattibilità clinica di un isolamento automatico e riproducibile delle cellule fetali circolanti ottenute in maniera non invasiva, da utilizzare per le analisi genetiche. Per questo motivo uno studio clinico di valutazione delle performance comincerà a breve su 1500 pazienti, reclutate in cinque diversi ospedali italiani. Obiettivi primari di questo studio saranno la valutazione delle performance in termini di sensibilità’ e specificità del metodo sviluppato, per il rilevamento delle aneuploidie fetali nelle gravidanze ad alto rischio. I risultati saranno comparati con i dati derivanti dalla diagnosi prenatale invasiva ottenuti dalle stesse donne che eseguiranno l’amniocentesi e la villocentesi. L’analisi comparativa determinerà i falsi postivi, falsi negativi, veri postivi e veri negativi del metodo sviluppato.
Current methods of prenatal diagnosis require fetal cells to be obtained through invasive procedures, risky for mother and fetus. The discovery of circulating fetal cells in 1979 and the possibility that these cells could be isolated from maternal blood during pregnancy was key to the development of alternative noninvasive approaches for identifying most fetal genetic abnormalities. All these methods result in a laborious, operator depending, time-consuming approach which until now it has not allowed to achieve a high and consistent purification of fetal cells. This project aims to develop a non-invasive method for the isolation of single fetal cells from maternal blood, for direct analysis of fetal chromosomes. The first part was dedicated to the research and testing of different specific markers for fetal cells enrichment and identification. Once optimized, the enrichment step was implemented to be automatic and integrated in a full workflow consisting of: pregnant women blood collection, positive magnetic enrichment, cell staining, single cell isolation and genetic analysis. As soon as the full workflow was standardized we started a clinical evaluation. To determine the success rate and number of trophoblast per sample, a total of 372 women were enrolled and stratified by gestational age at the time of blood collection. At least one fetal cell was isolated in 90.7% of the women sampled between 10-11 gestational weeks with an overall mean number of 3.5 recovered trophoblasts per patient. Furthermore, preliminary data from 131 women, showed a high concordance rate between isolated single trophoblastic cells and fetal karyotype for common trisomies and normal results deriving from gold standard invasive procedure. Overall, the results coming out from this study support the clinical feasibility of an automated and reproducible isolation of fetal cells for non-invasive prenatal genetic testing, well suited to the routine clinical practice. For this reason a clinical performance evaluation study will start soon, on 1500 patients, enrolled from five different Italian Hospital. Primary endpoints of the study will be the performance evaluation, in terms of sensitivity and specificity, of the developed workflow for fetal aneuploidies and segmental imbalances detection in a high-risk pregnancies population. Results will be compared with data resulting from invasive prenatal diagnosis for chromosomal abnormalities obtained on the same women presenting for hospital invasive procedure because classified from the physician as high risk pregnancy. The comparative analysis will determine the false positive, false negative, true positive, and true negative rates of the developed technology.

L’osteopetrosi autosomale recessiva (ARO) è un gruppo di malattie dovute a un difettoso funzionamento degli osteoclasti che preclude un rimodellamento osseo corretto. Nel 50% dei casi umani il difetto è dovuto ad una delezione nel gene Tcirg1. Il modello murino mutante oc/oc porta lo stesso difetto genetico e fenotipico umano. Nel lavoro di tesi si è dimostrato che gli epatociti fetali di 12.5 giorni di gestazione trapiantati in utero in feti mutati di 13.5 giorni di gestazione sono in grado di curare il fenotipo malato. Si è inoltre derivata una sottolinea di cellule staminali embrionali murine transgeniche per il costrutto plasmidico GOF18eGFP. Si vuole utilizzare la GFP sotto il controllo del promotore del gene Oct-4 come marcatore del livello di staminalità cellulare per microiniettare le ESC in blastocisti murine mutate oc/oc.
Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders due to defects that preclude normal function of osteoclasts. In half the cases, human ARO is due to mutations in the Tcirg1 gene. The oc/oc mutant mouse closely recapitulates human Tcirg1-dependent ARO. In ths work we demonstrate that in utero injection of allogenic fetal liver cells on 12.5 days into oc/oc fetuses at 13.5 day post coitum completely rescue the osteopetrotic phenotype. Moreover, an embryonic stem cells line transgenic for GOF18eGFP was produced. The goal is to use the GFP under the transcriptional control of the Oct-4 promoter as a marker of pluripotency of the ESC that are to microinject into oc/oc blastocysts.

L’osteopetrosi autosomale recessiva (ARO) è un gruppo di malattie dovute a un difettoso funzionamento degli osteoclasti che preclude un rimodellamento osseo corretto. Nel 50% dei casi umani il difetto è dovuto ad una delezione nel gene Tcirg1. Il modello murino mutante oc/oc porta lo stesso difetto genetico e fenotipico umano. Nel lavoro di tesi si è dimostrato che gli epatociti fetali di 12.5 giorni di gestazione trapiantati in utero in feti mutati di 13.5 giorni di gestazione sono in grado di curare il fenotipo malato. Si è inoltre derivata una sottolinea di cellule staminali embrionali murine transgeniche per il costrutto plasmidico GOF18eGFP. Si vuole utilizzare la GFP sotto il controllo del promotore del gene Oct-4 come marcatore del livello di staminalità cellulare per microiniettare le ESC in blastocisti murine mutate oc/oc.
Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders due to defects that preclude normal function of osteoclasts. In half the cases, human ARO is due to mutations in the Tcirg1 gene. The oc/oc mutant mouse closely recapitulates human Tcirg1-dependent ARO. In ths work we demonstrate that in utero injection of allogenic fetal liver cells on 12.5 days into oc/oc fetuses at 13.5 day post coitum completely rescue the osteopetrotic phenotype. Moreover, an embryonic stem cells line transgenic for GOF18eGFP was produced. The goal is to use the GFP under the transcriptional control of the Oct-4 promoter as a marker of pluripotency of the ESC that are to microinject into oc/oc blastocysts.

Negli ultimi anni si è assistito ad un crescente aumento nell’ambiente di sostanze che sono in grado di alterare il sistema endocrino poiché agiscono come l’ormone naturale estrogeno e per questo definite distruttori endocrini (EDs). Si ipotizza che l’esposizione agli EDs durante il periodo fetale e neo-natale sia la causa dell’insorgenza di numerosi disordini dell’apparato riproduttivo maschile come sterilità, cancro del testicolo, criptorchidismo e ipospadia che vengono definite con il termine unico di sindrome del testicolo disgenico (TDS). E’ importante studiare gli effetti degli estrogeni e degli xenoestrogeni, una classe di EDs, durante il periodo fetale ed in particolare durante lo sviluppo dell’apparto riproduttivo e conoscere i meccanismi, con cui tali sostanze esplicano i loro effetti. OBIETTIVI. Verificare l'espressione dei recettori degli estrogeni (ERs) nei precursori dei gameti adulti, ovvero nelle cellule germinali di topo denominate PGCs e analizzare i pathways molecolari attivati in queste cellule dagli estrogeni e dallo xenoestrogeno lindano. Inoltre si vuole verificare la presenza di ERs funzionali nelle cellule somatiche testicolari embrionali utilizzando costrutti ERE-luc e AP1-Luc per valutare l'attività estrogenica di xenoestrogeni. RISULTATI. Lo studio condotto in questa tesi mette in evidenza l'esistenza di pathways molecolari attivabili dall’estrogeno (E2) nelle gonadi embrionali di topo, in particolare, nel testicolo, sia nelle cellule germinali primordiali che nelle cellule somatiche. Abbiamo osservato che l’E2 è in grado di attivare, attraverso il recettore degli estrogeni ERα , importanti chinasi nelle PGCs (AKT, ERK1/2 e SRC) con effetti positivi sulla crescita e proliferazione di tali cellule. Si è osservato che il lindano, al contrario dell’E2, influenza negativamente la sopravvivenza di tali cellule attraverso una azione pro-apoptotica diretta, probabilmente derivante da effetti negativi sull’attività chinasica AKT. Inoltre abbiamo descritto per la prima volta l'esistenza di un recettore dell’estrogeno funzionale nelle cellule del Leydig testicolari durante lo stadio precoce dello sviluppo e messo a punto un test in vitro che può essere utilizzato per valutare l'attività estrogenica di xenoestrogeni direttamente sulle cellule del testicolo embrionale di mammiferi. CONCLUSIONI: Questi risultati rafforzano l’ipotesi dell’origine fetale della TDS. Numerosi studi hanno messo in evidenza gli effetti diretti degli estrogeni sul sistema endocrino, sull'espressione genica e su funzioni specifiche delle cellule somatiche testicolari embrionali, in particolare sulle cellule del Leydig, tuttavia tali studi sono carenti nelle cellule germinali. E’ importante conoscere i meccanismi di azione degli xenoestrogeni sullePGCs visto che attraverso esse viene trasmesso il genoma alle generazioni successive.
In the recent years the increased presence of human made compounds that mimic the action of estrogens termed endocrine disrupters (ED) in environment and in food and the exposure to these compounds during fetal and neonatal period has been hypotized to be the cause of the raise of disorders of male reproductive function, such a decrease of sperm count, increase in the incidence of testicular cancer and cryptorchidism and hypospadias termed Testicular Dysgenesis Syndrome (TDS). For these reason, it is important to know how the estrogens and xenoestrogens, a class of ED, act during the fetal development and to know the mechanism by which these compounds exert their effects. AIMS: To study the expression of estrogen receptors (ERs) in the embryonic precursors of the adult gametes termed PGCs and to analyze the existence in such cells of intracellular molecular pathways modulable by estrogens and xenoestrogen lindane. To verify the presence of functional ER-beta in embryonic testicular somatic cells using an ERE-luc and AP1-Luc assay and to evaluate estrogenic activity of putative EDs on mammalian embryonic testis. RESULTS: The data described in this thesis highlights the existence of functional estrogen-dependent pathways in embryonic mouse gonads in particular in testis, both in germ and somatic cells. We found that E2 is able to activate via ER-beta multiple intracellular signalling in PGCs and that the xenoestrogens, lindane affect the survival in such cells through a direct pro-apoptotic action likely resulting from its adverse effect on AKT activity. Othermore, we described for the first time the existence of a functional ERα pathway in putative Leydig cells from early stage of testis development and describe an in vitro assay that can be used to evaluate estrogenic activity of compounds on mammalian embryonic testis. CONCLUSIONS: These results support the notion of the TDS origin during early stages of testis development. While data are accumulating showing direct effect of estrogens and EDs on gene expression and specific functions of somatic cells of the embryonic testes, in particular Leydig cells, such results on germ cells are lacking and further studies are needed to investigate the effects of these compounds on embryonic germ cell function including epigenetic regulation.

Prenatal genetic diagnosis of monogenic diseases and chromosomal abnormalities is usually performed collecting fetal samples through villocentesis or amniocentesis. These invasive procedures are associated with 0.5-1% risk for the fetus. Due to it, in recent years, much effort has been made to develop non invasive prenatal diagnosis (NIPD). Two potential non invasive approaches involve the analysis of fetal cells and cell-free fetal DNA (cffDNA) found in the maternal circulation. The presence of fetal cells in the maternal circulation was first documented by 1893 when the fetal cells were found in pregnant women died for eclampsia. The Fetal erythroblasts (NRBC) from the maternal circulation have been considered the best potential target for NIPD because they can be detected early in pregnancy, are nucleated and have short life. The NRBC isolation from maternal circulation is still difficult because the number of NRBC is small and a fetal specific antibody is not currently available. Cff-DNA detected in the maternal circulation during pregnancy, could potentially offer an excellent method for early NIPD of the genetic status of a fetus. In a recent study has been shown that, during the pregnancy, the cffDNA is cleared from the maternal circulation, 16 min following delivery. Moreover, it has been demonstrated that cffDNA has a mean fractional concentration of about 10%. The goal of our project was to develop and validate a protocol for NIPD of monogenic diseases by two experimental procedures: the isolation and the molecular characterization of fetal NRBC, and the analysis of cffDNA in maternal plasma. We recruited 95 pregnant women between 6th and 14th week of gestation. In our study we have developed a protocol for fetal NRBC isolation from maternal blood that unfortunately require further refinements the isolation procedure. In parallel we have developed a protocol for non invasive fetal sexing from cffDNA. This method appears to be highly accurate showing 100% sensitivity and 96% specificity. In the next future we are planning to analyze cffDNA both for monogenic diseases and for the most frequent aneuploidies using massively parallel sequencing.

Chez la souris, l'ébauche de la rate foetale apparaît à jour embryonnaire 12,5 et contient des progéniteurs myéloides et érythrocytaires. À ce stade, la circulation sanguine est déjà établie et participe à la diversification du « pool » de cellules hématopoïétiques. Au cours de la vie foetale, les cellules hématopoïétiques de la rate prolifèrent et se différencient. Jusqu'à présent, le développement de la rate, son rôle sur la constitution du système hématopoïétique et les mécanismes qui participent à sa structuration ont été peu étudiés. Au cours de ma thèse, j'ai isolé les cellules stromales CD34 et CD34+ de la rate foetale qui pourraient contribuer activement au recrutement des cellules hématopoiétiques. J'ai étudié le contenu hématopoiétique de la rate foetale à différents stades de développement. J'ai caractérisé par des essais in vitro et in vivo une population CD4'nt qui se compose de progéniteurs T, NK, B et myéloides. Ces progéniteurs se différencieraient in situ et participeraient à l'activité hématopoiétique de la rate foetale. J'ai séparé ces progéniteurs en fonction de leur expression du récepteur Flt3 et du transgène RAG2-GFP et j'ai démontré que certains d'entre eux sont capables de se différencier en cellules inductrices des tissus lymphoïdes (ITL). Les ITL jouent un rôle majeur dans la génération des organes lymphoïdes. Afin d'évaluer la fonction des ITL dans la rate foetale, j'ai étudié leur positionnement et montré leur regroupement autour des vaisseaux sanguins. Grâce à un modèle de greffes spléniques, j'ai mis en évidence que les cellules B et les ITL occupent les mêmes régions. Ces résultats suggèrent que la position des futurs follicules lymphoïdes spléniques sont pré-établis par la régionalisation des ITL dans la rate foetale. Afin de déterminer les propriétés hématopoïétiques de l'environnement de la rate foetale, j'ai participé à l'établissement de 10 lignées de cellules stromales de rate foetales et étudié leurs capacités à soutenir la différenciation lymphoïde et myéloïde. Toutes ces lignées soutiennent l'engagement et la différentiation myéloïdes mais n'ont pas la même capacité à soutenir le développement lymphoïde. Cette diversité serait caractéristique de différentes niches de cellules stromales qui composent la rate foetale. Ces niches représenteraient des structures précoces des pulpes rouge et blanche
Mouse splenic anlagen appears at embryonic day 12. 5 (E12. 5) and contains erythroid and myeloid progenitors. At this stage, blood circulation is established and contributes to the hematopoietic pool diversification. During fetal life, the hematopoietic cells of the spleen proliferate and differentiate. Few studies have described the development of the spleen, the mechanisms that mediate the apparition of its structure and its role in the hematopoietic System constitution. During my thesis, I have isolated the fetal spleen CD34" and CD34+ stromal cell subsets that could actively contribute to the recruitment of hematopoietic cells. I have studied the fetal spleen hematopoietic content at different stages of development. I have characterized by in vitro and in vivo assays a CD4'm population that is composed of T, NK, B and myeloid progenitors. These progenitors could differentiate in situ and participate to the hematopoietic activity of the fetal spleen. I have further isolated these progenitors by considering their expression of Flt3 and RAG2-GFP transgene and demonstrated that some are able to give rise to the lymphoid tissue inducer (LTi) cells. These LTi cells play a major role in the lymphoid organ generation. To evaluate the LTi cell function in the fetal spleen, I have assessed their localization and shown their gathering around the blood vessels. Using spleen graft strategy, I have shown that LTi cells co-localize with B lymphocytes. These results suggest that the position of the future splenic lymphoid follicles is pre-established by LTi cells regionalization in the fetal spleen. To study the hematopoietic capacities of the fetal spleen environment, I have participated to the establishment of ten different fetal spleen stromal cell lines (FeSS) and determine their capacities to support the myeloid and lymphoid differentiation. All the FeSS promote the myeloid commitment and differentiation despite their heterogeneous ability to support the lymphoid development. This diversity could be related to the different niches of stromal cells that compose the fetal spleen. These different niches could be representative of the early red or white pulp structures

Nanoparticles are structures of different dimensions (1-100 nm) and composition (metal oxides, organic acid polymers, silica polymers) present in the environment as a consequence of natural processes (such as volcanic eruptions or dusts erosion) or anthropogenic activities (industrialization or pollution). In the last decades they have been intensely studied and engineered for industrial applications (as additive of food, cosmetics and building materials) and medical purposes, where they can be employed as drug carriers or imaging agents. Thanks to its abundance, cheapness and resistance to a variety of environmental perturbations, silicon dioxide (SiO2) is one of the most widely used materials in both industrial and biomedical fields in its amorphous form (contrary to crystalline silica that causes silicosis, a chronic pulmonary disease common in occupational categories largely exposed to silica crystals, such a miners and ceramic workers). Despite amorphous silica is considered much less dangerous than crystalline silica, recent evidences have shown cytotoxic and pro-inflammatory potential in in vitro and animal models. To shed more light on this topic, in the first part of my PhD work I have characterized amorphous silica toxicity and inflammatory effects in primary human monocytes and macrophages (myeloid professional phagocytic cells) and on primary human lymphocytes and HeLa cells (lymphoid and epithelial non-phagocytic cells), using as nanoparticles model the commercial non labeled Ludox TM40 (29 nm Ø) and the fluorescein labeled Stöber (35 nm Ø). In particular, the influence of serum and the possible mechanisms determining silica nanoparticles (SiO2-NPs) effects have been investigated. I have found that SiO2-NPs toxicity (evaluated as mitochondrial dysfunction and plasma membrane permeabilization) was stronger in phagocytes (LD50 after 18 h exposure 40 µg/ml) and, in these cells, associated to the production of the three main inflammatory cytokines (IL-1 beta, TNF- alpha and IL-6). On the contrary, non phagocytic cells were much more resistant (LD50 after 18 h exposure 300-500 µg/ml) and, curiously, HeLa cells were subjected apoptosis after SiO2-NPs treatment (while monocytes, macrophages and lymphocytes became necrotic). Cytofluorimetric and confocal microscopy analysis have shown that SiO2-NPs were engulfed in acidic compartments (phagolysosomes) in monocytes and macrophages, while they mostly localized onto plasma membrane in HeLa cells. This suggested that the higher sensitivity of the two phagocytic models could be due to the more efficient internalization of the particles, followed by lysosomal rupture (as indicated by experiments showing the decrease of the fluorescence associated to the lysosomotropic agent Lysotracker upon cellular exposure to high NPs doses) and the consequent liberation of lysosomal proteases (as reported in literature for crystalline silica). To investigate if acidic lysosomal pH could influence SiO2-NPs cytotoxicity in phagocytes, cells were treated with silica in the absence or in the presence of two pH neutralizing agents (NH4Cl and Bafilomycin AI), resulting in a significant protective effect in monocytes and in a negligible protection in macrophages. As mentioned above, we found that in myeloid cells SiO2-NPs induced an inflammatory response (more pronounced in monocytes), starting in the correspondence of the beginning of cytotoxicity, reaching a peak and decreasing at high NPs doses because of the strong and anticipated cellular death. In particular, IL-1b was the cytokine most represented in both cellular types, while TNF-alpha and IL-6 levels were lowers. Moreover, in monocytes (and, in less degree in macrophages) IL-1beta production was synergized by the co-stimulation with silica and lypopolisaccharide (LPS). Since crystalline silica is known to activate the NLRP3 inflammasome (a cytosolic multiprotein complex responsible of the production of some inflammatory cytokines, primarily IL-1beta) we investigated NLRP3 activation by amorphous SiO2-NPs in our myeloid cellular models. We found that monocytes and macrophages treatment with SiO2-NPs increased pro-IL1beta levels, and that its conversion into mature IL-1beta involved caspase 1 activation, intracellular ATP release and subsequent binding to ATP receptor P2X7. Interestingly, P2X7 blockage did not affect SiO2-NPs induced cellular death in both cells, and caspase 1 inhibition did not reduce SiO2-NPs toxicity in macrophages but showed a protective effect in monocytes, suggesting that amorphous silica might induce pyroptosis (a caspase 1 dependent cellular death) in these cells. Afterwards, I have studied how serum could modulate SiO2-NPs toxicity and pro inflammatory effects. In the presence of increasing FCS amount both SiO2-NPs cytotoxicity threshold and LD50 were shifted to higher NPs doses, indicating a serum protective action (more pronounced in non-phagocytic cells in comparison to phagocytes). In parallel, also inflammatory cytokines production in myeloid cells occurred at higher NPs concentrations with the increment of FCS percentage. To investigate if the protective serum effect reflected a modification in NPs cellular association I have performed experiments with the fluorescent Stöber NPs, finding that the presence of serum strongly decreased NPs cellular binding in lymphocytes and HeLa, with a moderate reduction in monocytes and macrophages. This result was in accord with the stronger serum protection in non phagocytic cells, and consolidated the hypothesis that SiO2-NPs toxicity depends on their interaction level with cells. Also SiO2-NPs cellular localization was affected by different serum concentrations. In particular, I have found that in the absence of serum SiO2-NPs mostly localized onto the plasma membrane in monocytes and HeLa, while in macrophages they were still internalized into phagolysosomes, even if with a lower efficiency. Considering this different NPs sub-cellular localization, I have investigated if the protective effect of NH4Cl, Bafilomycin AI and caspase 1 inhibitor observed in monocytes in standard culture conditions (10% FCS) was maintained also in the absence of serum. Interestingly, neither acidic compartments neutralization nor caspase 1 blockage were able to reduce SiO2-NPs toxicity in serum free conditions, suggesting that monocytes cellular death mechanism was different with or without serum. In the second part of my PhD work I have studied several aspects of nanoparticles interaction with plasma and serum proteins, since this is a very important point in nanomaterials biomedical applications. Indeed, when nanoparticles (functioning as drug-gene vectors or imaging agents) are introduced in the human body (primary, into the bloodstream), they are rapidly coated by a series of specific proteins, forming the so called “ nanoparticles corona” and mediating cellular response to nanomaterial. This problematic was approached by using Ludox TM40 as nanoparticles model (to maintain the continuity with the previous characterization work), and performing a comparative analysis between fetal calf serum and human plasma. I have found that the main proteins adsorbed to NPs surface were plasminogen, albumin, apolipoprotein AI (ApoAI), hemoglobin and apolipoprotein AII (ApoAII) from FCS, and immunoglobulins (IgG), histidine rich glycoprotein (HRG), albumin, apolipoprotein AI, apolipoprotein AII and apolipoprotein CIII (ApoCIII) from human plasma. In both situations, albumin (the most abundant plasma/serum protein) was the principal polypeptide bound to NPs at very low serum/plasma concentrations, while at greater serum/plasma doses it was displaced by less plentiful proteins (over all, apolipoproteins), indicating an higher affinity of these latter for SiO2-NPs surface. Congruent with this, NPs firstly absorbed plasminogen, apolipoproteins and hemoglobin, while once these proteins were depleted from serum albumin started to bind NPs surface proportionally to NPs dose. I have also found that the serum proteins pattern associated to NPs surface was not rearranged from a neutral environment (representative of cytosol) to an acidic environment (representative of endo-lysosomes). Finally, I have investigated how single plasma opsonines could influence SiO2-NPs biological activity, finding that IgG and HRG did not protect cells against NPs toxicity, while albumin and, over all, high density lipoproteins (the complexes containing ApoAI, ApoAII and ApoCIII) strongly reduced NPs adverse effects by inhibiting NPs cellular association
Le nanoparticelle sono strutture di diverse dimensioni (1-100 nm) e varia composizione (ossidi metallici, polimeri di silice, polimeri di acidi organici), presenti nell’ambiente come conseguenza di processi naturali (eruzioni vulcaniche, erosione di rocce) o antropogenici (inquinamento, attività industriale). Negli ultimi decenni esse sono state intensamente studiate e ingegnerizzate a scopo industriale (come additivi di cibi, cosmetici e materiali utilizzati nell’edilizia) e nell’ambito biomedico, in cui possono essere impiegate come vettori per farmaci o agenti di imaging. Grazie alla sua abbondanza, economicità e resistenza, il biossido di silicio (SiO2) è, nella sua forma amorfa, uno dei materiali maggiormente usati sia in ambito industriale che biomedico (contrariamente alla forma cristallina che provoca l’insorgenza della silicosi, una malattia polmonare cronica molto diffusa nei minatori, lavoratori di ceramica e altre categorie occupazionali quotidianamente esposte a cristalli/fibre di silicio). Nonostante la silice amorfa sia considerata molto meno pericolosa di quella cristallina, studi recenti condotti in vitro e su modelli animali hanno messo in luce un potenziale citotossico e pro infiammatorio. Per chiarificare questo aspetto, nella prima parte del mio dottorato ho caratterizzato la citotossicità e l’induzione di una risposta pro infiammatoria di nanoparticelle di silice amorfa (la variante commerciale non fluorescente Ludox TM40 e la variante fluorescente Stöber) su due modelli di cellula fagocitica (monociti e macrofagi primari umani) e in due modelli di cellula non fagocitica (linfociti primari umani e la linea stabile epiteliale HeLa). In particolare, mi sono concentrata sui possibili meccanismi coinvolti nell’azione delle nanoparticelle e su come il siero possa influenzarli. Un primo risultato ha indicato una maggior tossicità delle nanoparticelle di silice (valutata in termini di disfunzione mitocondriale e permeabilizzazione della membrana) nelle cellule fagocitiche, associata anche alla produzione delle tre principali citochine pro infiammatorie (IL-1beta, TNF-alfa and IL-6). Più in dettaglio, la dose di nanoparticelle in grado di uccidere il 50% delle cellule (LD50) dopo un trattamento di 18 h è risultata essere di 40 µg/ml, mentre le cellule non fagocitiche hanno mostrato una maggior resistenza alle nanoparticelle (LD50 300-500 µg/ml). Analizzando il tipo di morte indotta dalle nanoparticelle di silice, le cellule HeLa hanno mostrato un fenotipo apoptotico, mentre i monociti, macrofagi e linfociti sono risultati andare incontro a necrosi. Da una valutazione al citofluorimetro e al microscopio confocale dell’associazione e della localizzazione cellulare delle nanoparticelle è emerso che queste venivano internalizzate in compartimenti acidi nelle due cellule fagocitiche, mentre nelle cellule HeLa rimanevano legate alla membrana plasmatica. Questo risultato ha suggerito che la maggior sensibilità dei fagociti fosse dovuta a una maggior captazione delle nanoparticelle che, una volta accumulate all’interno di endo-lisosomi, potessero provocarne la rottura con la conseguente liberazione di enzimi litici (proteasi, idrolasi, lipasi) in grado di danneggiare la cellula. A questo proposito, è stato valutato il contributo dell’ambiente acido degli endo-lisosomi alla tossicità delle nanoparticelle di silice nei fagociti trattando le cellule in presenza o in assenza di due agenti neutralizzanti (NH4Cl o Bafilomicina AI), ottenendo una diminuzione della citotossicità della silice nei monociti e solo un lieve effetto nei macrofagi. Come anticipato precedentemente, le nanoparticelle di silice si sono mostrate in grado di indurre una risposta infiammatoria (più evidente nei monociti) caratterizzata da un’iniziale fase di latenza fino al raggiungimento della soglia di tossicità, un picco centrale e una fase finale decrescente (in corrispondenza delle dosi più alte di nanoparticelle) a causa della forte e anticipata morte cellulare. In particolare, l’ IL-1beta è risultata essere la citochina prodotta più abbondantemente, seguita dal TNF-alfa e dall’IL-6; inoltre, in copresenza di nanoparticelle e LPS essa veniva secreta in modo sinergico. Dal momento che la silice cristallina è in grado di attivare l’inflammasoma NLRP3 (un complesso multi proteico citosolico responsabile della produzione di alcune citochine pro infiammatorie, prima fra tutte l’ IL-1beta una parte del lavoro di tesi è stata dedicata allo studio dell’attivazione di NLRP3 da parte di nanoparticelle di silice amorfa nei nostri due modelli di cellula fagocitica. Inizialmente è stata evidenziata sia in monociti che in macrofagi la capacità delle nanoparticelle di silice amorfa di aumentare i livelli di pro IL1-beta e stimolarne la conversione nella forma matura IL-1beta tramite un processo dipendente dall’attivazione della caspasi 1, della secrezione di ATP ed dal successivo legame di ATP al suo recettore P2X7. Inoltre, a seguito del blocco di P2X7 con uno specifico inibitore la mortalità indotta dalle nanoparticelle di silice non ha subito variazioni sia nei monociti che nei macrofagi, mentre i monociti hanno mostrato una maggior resistenza alle nanoparticelle in presenza di un inibitore della caspasi 1, segno di una possibile morte per piroptosi causata dalle nanoparticelle in queste cellule. Un altro aspetto importante presentato in questa tesi di dottorato riguarda l’influenza del siero (FCS) sugli effetti citotossici e pro infiammatori indotti dalle nanoparticelle di silice amorfa. Innanzitutto, all’aumentare della concentrazione del siero sia la soglia di tossicità sia l’LD50 delle nanoparticelle di silice sono risultate spostate verso valori più alti, indicando un effetto protettivo dell’ FCS (più evidente nei non fagociti rispetto ai fagociti), cosi come la produzione di citochine pro infiammatorie nei monociti e nei macrofagi. Per capire se questo spostamento fosse dovuto a una diversa associazione delle nanoparticelle alle cellule sono stati fatti esperimenti con le nanoparticelle fluorescenti Stöber, che hanno evidenziato come la presenza di siero fosse in grado di diminuire il legame fra la nanoparticelle e le cellule, in particolare nel caso dei linfociti e delle HeLa. Questo risultato è in accordo con la precedente osservazione di un maggior effetto protettivo del siero nei non fagociti, e rafforza l’ipotesi che la tossicità delle nanoparticelle sia in qualche modo legata al loro livello di interazione con le cellule. Inoltre, anche la localizzazione intracellulare delle nanoparticelle è risultata essere influenzata dalla concentrazione di siero. In particolare, in assenza di siero le nanoparticelle erano prevalentemente distribuite sulla membrana cellulare nei monociti e nelle HeLa, mentre nei macrofagi venivano internalizzate in fago-lisosomi, anche se meno efficientemente che con 10% FCS. Vista la diversa localizzazione subcellulare nelle due diverse condizioni di siero, ci si è chiesti se l’effetto protettivo dell’ NH4CL, Bafilomicina AI e dell’inibitore della caspasi 1 osservato nei monociti nelle condizioni di coltura standard (10% FCS) venisse mantenuto anche in assenza di siero. Né la neutralizzazione dei compartimenti acidi né l’inibizione della caspasi 1 si sono dimostrati efficaci nel prevenire la tossicità, indicando che nei monociti il meccanismo di morte cellulare fosse diverso in assenza o in presenza di siero. Nella seconda parte della mia tesi di dottorato ho analizzato diversi aspetti dell’interazione delle nanoparticelle con le proteine del plasma e del siero, essendo questo un aspetto cruciale nell’applicazione dei nanomateriali in campo biomedico. Infatti, nanoparticelle introdotte nell’organismo (in particolare del circolo sanguigno come vettori per farmaci o agenti d’immagine) vengono rapidamente rivestite da una serie di proteine (costituenti la cosiddetta “corona di proteine”) in grado di mediare l’interazione cellula-nanoparticella. Questa problematica è stata affrontata utilizzando come nanoparticelle modello le Ludox TM40 (per mantenere la continuità con la caratterizzazione fatta in precedenza) ed eseguendo esperimenti in parallelo con il siero fetale bovino ed il plasma umano. Le principali proteine del siero bovino legate alle nanoparticelle di silice sono risultate essere il plasminogeno, l’albumina, le apolipoproteine AI e AII e l’emoglobina, mentre quelle del plasma umano le immunoglobuline, l’histidine rich glycoprotein, l’albumina e le apolipoproteine AI, AII e CIII. Il pattern di proteine adsorbite alle nanoparticelle di silice ha evidenziato che a basse concentrazioni di siero/plasma la principale proteina legata era l’albumina (la proteina più abbondante del siero/plasma), mentre all’aumentare della concentrazione di siero/plasma essa veniva “spiazzata” da proteine meno abbondanti (in primis dalle apolipoproteine), suggerendo una maggiore affinità di queste ultime per la superficie di nanoparticelle di silice amorfa. In accordo con questa ipotesi, le nanoparticelle inizialmente legavano il plasminogeno, l’emoglobina e le apolipoproteine, mentre solo quando queste proteine venivano esaurite dal siero (in presenza di alte dosi di nanoparticelle) iniziava a legarsi l’albumina, proporzionalmente alla quantità di nanoparticelle presenti. Dall’analisi del pattern di proteine legate alla silice in presenza di differenti pH è emerso che esso non subiva variazioni rilevanti in presenza di un ambiente neutro (rappresentativo del citoplasma) o di un ambiente acido (rappresentativo degli endo-lisosomi). Infine, esperimenti preliminari su come le singole proteine della corona potessero influenzare l’attività biologica delle nanoparticelle hanno indicato né le immunoglobuline né l’HRG erano in grado di diminuire gli effetti citotossici delle nanoparticelle, mentre l’albumina e, in particolare, le HDL erano fortemente protettive, riducendo l’associazione delle nanoparticelle alle cellule

The fetal cell microchimerism (FCM) is defined as the persistence for decades after pregnancy of fetal cells in maternal circulation and organs without any apparent form of rejection. The FCM is a common occurrence during pregnancy and, in fact, was detected at a frequency of 20-75% in the peripheral blood of women with sons and of 40-100% in the tissues of patients with malignant or benign diseases. Nevertheless, the physiological consequences of this phenomenon remain to be clarified in both healthy women and in those affected by carcinoma. There are conflicting data about the pathogenic role of FCM in autoimmune diseases and in tumors. In short, most of the studies provides that the FCM can play a pathogenic role in autoimmune diseases and conversely a protective role for neoplastic diseases. In this context, our group has recently demonstrated a possible role of FCM in the damage-induced thyroid cancer and in the recovery or protection of the affected tissue. In particular, male cells were found in a high proportion of women than had a male pregnancy before the diagnosis of cancer. These cells were found to be significantly represented in contralateral cancer tissue compared to healthy tissue. To extend in the peripheral blood our knowledge of the FCM in thyroid cancer and to explore the possible relationship between the presence of microchimerism and the development of this tumor were enrolled 106 women with a previous male pregnancy, including 57 patients with papillary carcinoma thyroid cancer (PTC) diagnosed after pregnancy and 49 healthy controls. At the level of peripheral blood FCM was found less frequently in women with PTC compared with healthy controls. These findings suggest a protective role of microchimerism on the development of cancer. However additional data are needed to know in depth the role of FCM is the development of cancer and autoimmune diseases.

Magister Scientiae (Medical Bioscience) - MSc(MBS)
Several studies conducted in laboratories at the University of the Western Cape has demonstrated an interference with the parenchymal lung tissue of the offspring when exposed to nicotine (smoking cigarettes and/or Nicotine Replacement Therapy [NRT]), maternally i.e. during gestation and lactation. This in turn, decreases the amount of air sacs (alveolar number) resulting in a reduced surface area available for efficient gas exchange in the offspring. Since the foetus and offspring are only exposed to nicotine during gestation and lactation, emphysema- like lesions appear to develop after nicotine withdrawal in the foetus. It has been proposed that during lung development in utero, a change in the "program" that controls the maintenance of lung integrity will occur in the long term due to the initial maternal nicotine exposure. Therefore, animals that were exposed to maternal nicotine resemble lungs that have undergone rapid, premature aging caused by cellular senescence. Furthermore, energy metabolism and structural changes in the glycolytic pathways appear irreversibly slower compared to animals that were not exposed to nicotine via the mother during gestation and lactation, resulting in a reduction in the anti-oxidant capacity of lung development. Previous studies have also shown that strong anti-oxidants supplemented by smoking mothers during gestation and lactation could possibly resist change in the "program" which controls lung development and integrity of the offspring in the long term. Lycopene – as a strong anti-oxidant supplementation have shown to decrease the alveolar volume and increase the alveolar surface area for better gas exchange after the offspring has been exposed to maternal nicotine. In this study I have treated pregnant wistar rats with nicotine, tomato juice (containing lycopene among other phytonutrients), and a combination of nicotine and tomato juice during gestation, to determine various changes in the lung structure and signs of premature aging in the lungs of the offspring. I have also performed various staining techniques such as H&E, connective tissue and β- galactosidase staining which indicated whether maternal nicotine exposure indeed induced premature cellular senescence in the lungs of the offspring.
National Research Foundation

Childhood Leukaemia accounts for nearly one in three of all childhood cancers, with approximately 400 cases diagnosed in the UK every year. Initial genetic lesions in paediatric and infant B cell leukaemias occur in utero in fetal haematopoietic progenitors. Despite the importance of understanding fetal B cell lymphopoiesis in relation to the aetiology of leukaemia, fetal B cell development remains poorly characterised. We hypothesised that by understanding and characterising murine fetal B cell lymphopoiesis, we would gain insights into the development of childhood leukaemias. In chapter III of this thesis I show that the first immune restricted cells with B cell potential in the mouse emerge in the E9.5 vascular plexus of the yolk sac. Subsequently, in chapter IV, I use a Mb1-cre fate mapping approach to show that progenitors which are entirely restricted to the B cell lineage can be detected as early as E12.5 in the fetal liver (FL), prior to the cell surface expression of CD19. In chapter V, I investigate B cell progenitors in the E14.5 FL, applying an advanced B cell staging originally developed for the adult bone marrow, modified to include the key lymphoid cytokine receptors IL-7Ra, KIT and FLT3. I identify and functionally and molecularly characterise a rare CD19+ B cell progenitor that expresses FLT3 and peaks in frequency in late gestation embryos. I further show that FLT3+ CD19+ adult Pro B cells demonstrate an increased engraftment when transplanted into recipient mice compared to CD19+FLT3- Pro B cells. Finally in chapter VI, I apply our new understanding of fetal B cell lymphopoiesis to characterise B cell progenitors in a mouse that expresses the TEL-AML1 translocation, an oncogene associated with 25% of paediatric B cell leukaemias. We find that the novel FLT3+ CD19+ ProB cell progenitor identified here is expanded in the fetal liver of E14.5 TEL-AML1 embryos, giving a new developmental insight into the functional impact of this oncogene.

Dans la présente étude, nous avons étudié la possibilité de différencier les cellules souches du liquide amniotique humain (hAFSC) et murin (mAFSC) vers la voie hématopoïétique à la fois in vitro et in vivo. Nous avons de manière reproductible réalisé une différenciation érythroïde par culture des hAFSCs en corps embryoïdes (EB). Plus de 70% des cellules constituant les EB coexprimaient des marqueurs erythroïdes. De plus, 3 mois après l'injection de hAFSC à des souris NOD/SCID irradiées, nous avons pu détecter dans la rate et la moelle osseuse des receveurs des érythrocytes humains. Nous avons ainsi comparé le potentiel hématopoïétique des mAFKL, des mAmKL aux KL issues du foie foetal (mFLKL), qui constituent la source principale de cellules souches hématopoïétiques à ce stade de développement. In vivo, nous avons retrouvé dans le sang de souris déficientes pour RAG1, des cellules appartenant aux trois lignées hématopoïétiques (lymphoïde, erythroïdes et myéloïdes) 4 semaines seulement après la greffe de mAFKL et mAmKL. Analysées quatre mois plus tard, les souris greffées présentent des lymphocytes, des érythrocytes et des cellules myéloïdes provenant des mAFKL et mAmKL dans tous les organes hématopoïétiques. Le succès de transplantations secondaires a confirmé que les mAFKL et mAmKL comprennent des progéniteurs hématopoïétiques capables d'auto-renouvellement, ce qui correspond à la définition d'une cellule souche hématopoïétique
In the present study we investigated the possibility of differentiating AFS cells towards the hematopoietic pathway. We achieved a reproducible erythroid differentiation by culturing hAFSCs as embryoid bodies (EBs) under serum free conditions with haematopoietic cytokines. Furthermore, human erythrocytes (human CD235a) were isolated from bone marrow and spleen of sublethally irradiated NOD/SCID mice at 3 months after the injection of hAFSCs. We compared the hematopoietic potential of mAFKL and mAmKL to Fetal Liver KL, the main source of fetal HSC. When cultivated immediatly after their sorting, freshly isolated murine AFKL and AmKL cells gave rise to all the different hematopoietic lineages both in vitro and in vivo. Experiments with freshly isolated hAFKL gave good results in the in vitro assays being able to give rise to erythroid, myeloid and lymphoid lineages, but failed to reconstitute the hematopoietic system in irradiated NOD/SCID mice, probably due to the poor amount of cells injected. This is the first report demonstrating that AFKL and AmKL do have an haematopoietic potential, supporting the idea that AF and Am may be an excellent source for therapeutic application

Skeletal muscle (SkM) possesses an impressive ability to regenerate in response to injury or chronic disease. This regenerative capacity is attributed to its resident mononuclear myogenic progenitors. Previous studies have identified several types of myogenic progenitors within SkM, some of which are isolated by fluorescence activated cell sorting (FACS) using cell surface markers. Studies in our laboratory have identified melanoma cell adhesion molecule (MCAM) as a cell surface marker expressed by myogenic progenitors in human fetal SkM. However, the relationship between MCAM expression and the degree of myogenic commitment of distinct MCAM+ populations has not been elucidated. In the present study, subpopulations of MCAM+ cells were purified by FACS on the basis of Hoechst 33342 dye uptake. Specifically, MCAM+ side population (SP) was isolated by Hoechst exclusion and MCAM+ main population (MP) on Hoechst incorporation. Sorted populations were first optimized for growth in vitro since SkM SP cells are difficult to maintain in culture. In particular, Invitrogen’s StemPro® MSC SFM medium was found to support propagation of human fetal SkM SP cells with minimal differentiation. Following this optimization, sorted populations were assessed for expression of myogenic markers before and after propagation and then for fusion potential in vitro and engraftment potential in vivo. The MCAM+ subpopulations were found to express myogenic markers to a significantly greater extent than MCAM- subpopulations. Furthermore, the MCAM+ subpopulations fused robustly into myotubes in vitro whereas the MCAM- subpopulations did not. Interestingly, the MCAM+ SP population exhibited the highest fusion potential in vitro and was the only MCAM+ subpopulation to engraft into dystrophic muscle in vivo following propagation. These results indicate that MCAM is associated with myogenicity and can be used to prospectively isolate a pure myogenic fraction from human fetal SkM tissue. Moreover, the MCAM+ SP retain its myogenic potential to a greater extent than MCAM+ MP after propagation. This suggests that the MCAM+ SP fraction contains a higher percentage of early myogenic progenitors compared to the MCAM+ MP fraction. Additional studies on MCAM-expressing populations in human fetal SkM may elucidate a potent population for use in cell-based therapeutic strategies for treating muscle diseases.

The Wharton’s Jelly (WJ) of human umbilical cord (HU) contains multipotent stem cells (WJ-MSC) which express mesenchymal markers but not hematopoietic markers. WJ-MSC are increasingly being tested for use in stem cell therapy, with intravenous delivery being the preferred route. Fetal stem cells from embryonic germ layers are present in maternal blood and can home to damaged maternal tissues. This study investigates how WJ-MSC cross the fetal and adult endothelial barriers; including the cellular and molecular mechanisms employed. WJ-MSC were isolated from HU (n=27) which were taken from normal term pregnancies after elective Caesarean section. Flow cytometry and immunofluorescence were used to check presence/absenc of mesenchymal versus haematopoietic markers. Cells were induced to become adipocytes, chondrocytes and osteocytes by using specific induction medium. Isolated WJ-MSC were added after labelling with PKH26 to confluent monolayers of isolated human umbilical vein endothelial cells (HUVEC) or commercially bought human uterine microvascular endothelial cells (HUtMEC) at a 1:5 ratio. Cell-cell interactions were monitored with real time microscopy for 24 to 40h. Fluorescence and confocal scanning microscopy, after vascular endothelial (VE) cadherin immunocytochemistry were used for detailed analysis of VE-cadherin junctional occupancy and spatio-temporal location of stem cells. Tyrosine phosphorylation status of VE-cadherin, whether at Tyr685 or Tyr731, at different time points were investigated by immunoblotting whilst levels of vascular endothelial growth factor (VEGF) in the conditional media (CM) were measured by ELISA. Three different isolates were tested, with 3 experimental repeats for all expermints. Statistical analyses were performed with ANOVA (One or Two way). Cells (>95%) from each passage were positive for the mesenchymal markers CD 29, CD 105, CD 90, CD 73 and CD 44. <2% cells showed positivity to the haematopoietic markers CD 34, HLA-DR, CD 14, CD 19 and CD 45. WJ-MSC differentiated into adipocytes, osteocytes and chondrocytes. WJ-MSC displayed exploratory behaviour for a minimum of 30 min on HUtMEC or 60 min on HUVEC with interrogation of paracellular openings before crossing rather than replacing endothelial cells. By 2h, half were found at sub-endothelial positions, with a majority reaching this within 16-22h. There was accompanying loss of junctional VE-cadherin (64.9 + 3.7 %; p<0.001 in HUVEC; 63 + 4.6%; p< 0.001 in HUtMEC) in the endothelial monolayers followed by a return at 16h and increased continuity by 22h (p<0.01 in HUVEC; p<0.001 in HUtMEC). Junctional disruptions were found close to overlying or migrating WJ-MSC. Confocal microscopy confirmed paracellular extravasation. VE-cadherin protein levels matched controls in the early hours (0-2h) and increased after 22h co-culture in both fetal and uterine endothelium. VE-cadherin showed a 2-fold increase in phosphorylation at Tyr685 from 30 min to 2h. P-Tyr731 remained unchanged, similar to untreated endothelial layers, then decreased at 2h and 22h. VEGF levels in WJ-MSC – HUtMEC co-culture supernatants was highest at 2h (88 + 3 pg/ml) and decreased by 22h, reaching negligible levels by 48h. Anti-VEGF blocked Tyr685 phosphorylation but did not affect the decrease in P-Tyr731; this was accompanied by a 25% decrease in transmigration of cells in the first two hours and a 43% decrease in total by 22h. in WJ-MSC – HUtMEC co-cultures. However, in HUVEC-WJ-MSC co-cultures, no VEGF were detected and anti-VEGF did not block Tyr685 phosphorylation and Tyr731 de-phosphorylation. WJ-MSC from term umbilical cords can be easily isolated and expanded in culture. They retain mesenchymal stem cell properties for the passages tested (up to P5) making them a valuable model for studies into mechanisms underlying extravasation. The data obtained suggest that WJ-MSC can influence expression of VE-cadherin, with perturbation during transmigration followed by upregulation and repair once the adlumenal side is reached. There was a similarity in the cellular and molecular mechanisms employed by WJ-MSC in their paracelluar migration across fetal and uterine endothelium, although VEGF may not be the key player in HUVEC interactions. For both endothelial types, WJ-MSC appear to induce phosphorylation events linked with paracellular permeability and de-phosphorylation events normally associated with leukocyte extravasation. The data from the uterine endothelial investigations suggests that fetal stem cells are able to influence paracellular junctional dynamics and strengthens the growing hypothesis that they may also play a role in re-modelling the uterine circulation for fetal advantage. The extra-embryonic WJ-MSC holds the promise of use in restoring junctional maturity and vascular repair in future therapeutic applications.

Abstract : Current prenatal diagnosis depends on invasive procedures and is thus offered only to high-risk pregnancies. Development of non-invasive prenatal diagnosis (NIPD) would change the risk-benefit ratio and make it likely that more women would benefit from prenatal testing. Scientists have documented the presence of rare fetal cells in maternal blood and envisioned targeting them with specific markers and their use in NIPD. Considering their extremely low frequency in maternal blood, fetal cells have been difficult to retrieve and use in clinical practice. Therefore, there is a pressing need for systematic sequential studies to evaluate their feasibility in NIPD. Generally, detection of rare cells within a large cell population carries great potentialities for the prospects of cancer management and NIPD. Manual scanning is very cumbersome and time-consuming Therefore; the first part of our project was, dedicated to the optimization of an effective strategy to evaluate retrieval of rare cells. We have developed a way of distributing a controlled number of target cells among hundreds of thousands of other cells on microscope slides. This strategy allows the precise evaluation of the retrieval of rare events and the comparizon of the efficacy of different techniques and enrichment approaches by knowing the definite number and locations of target cells on the slides. Furthermore, it allows the evaluation of hybridization of missed events. We have also developed a robust custom-made detection algorithm for rare cells using the MetaSystems automated platform and have used this strategy in the validation of manual and automatic scanning of 60 slides with a pre-defined number of rare male cells among a pure population of female cells using XY-FISH. Consequently, we tested the developed classifier for the detection of real fetal cells from maternal blood in both normal and aneuploid pregnancies with Down syndrome. We further evaluated the number of fetal cells with different methods of enrichments in the first and second trimesters. The data collected confirmed the early presence of fetal cells in all of the pregnancies tested and their frequencies were higher in cases of aneuploidies. Fetal cells are in a state of dynamic change throughout the pregnancy. Higher numbers of these cells can be obtained by optimizing the harvest time and methods of enrichment. We found that automatic scanning is more sensitive and reliable than manual detection. Furthermore, it alleviates the burden of scanning large numbers of cells and thus is more suitable for clinical application. We also demonstrated the feasibility of using rare cells in NIPD. Five microdissected amniotic fetal cells from 26 cases of normal and aneuploid pregnancies were quite enough to provide accurate NIPD through using whole genome amplification coupled with QF-PCR. Our findings laid the ground for the use of rare fetal cells in maternal blood for NIPD. // Résumé : Le diagnostic prénatal résulte encore aujourd’hui de procédures invasives, qui présentent des risques pour la grossesse. Le développement du diagnostic prénatal non-invasif (DPNI) changerait le rapport risque : bénéfice, rendant le diagnostic prénatal plus intéressant pour les femmes enceintes. Plusieurs chercheurs ont montré la présence de cellules fœtales dans le sang maternel et des travaux ont été entrepris afin de les cibler et de les utiliser éventuellement en DPNI. Toutefois, la faible concentration des cellules fœtales dans le sang maternel réduit les possibilités d’isolement ainsi que celles de leur utilisation en clinique. Un autre aspect technique du DPNI, le balayage manuel, est très laborieux, surtout en terme de temps technique. Il y a donc un besoin certain pour des études approfondies afin d’évaluer et d’améliorer la faisabilité du DPNI. La détection d’évènements rares dans une grande population cellulaire offre un potentiel pour le diagnostic en oncologie mais aussi en diagnostic prénatal. Dans cette thèse, la première étude était dédiée à l’optimisation d’une stratégie pour détecter les cellules rares. Nous avons développé une méthode d’étalement sur lame d’un nombre précis de cellules cibles parmi des centaines de milliers de cellules. Cette stratégie a permis d’évaluer le taux de détection d’évènements rares et de comparer l’efficacité des techniques d’enrichissement en connaissant le nombre exact et la localisation de cellules cibles sur les lames. De plus, il a été possible d’évaluer les problèmes d’hybridation des évènements manqués. Nous avons, par la suite, développé un algorithme robuste pour la détection de cellules rares en utilisant la plateforme de microscopie automatisée MetaSystems et utilisé cette approche dans la validation des balayages manuel et automatique d’un nombre précis de cellules mâles parmi une large population de cellules femelles marquées avec la technique FISH. Nous avons testé ce classificateur avec des échantillons de sang de femmes enceintes de grossesses normales et aneuploïdes et évalué la fréquence de cellules fœtales isolées par différentes méthodes d’enrichissement au cours des premier et second trimestres de grossesse. Les données accumulées ont confirmé la présence de cellules fœtales chez toutes les grossesses et leur fréquence plus élevée dans les grossesses aneuploïdes. Le nombre de cellules fœtales est dynamique tout au long de la grossesse. De plus, un nombre plus élevé de cellules fœtales peut être obtenu en optimisant le moment du prélèvement et les méthodes d’enrichissement. De plus, le balayage automatique s’est avéré plus sensible et constant que le balayage manuel, ce qui permet de balayer un grand nombre de cellules et devient plus approprié pour une application clinique. Nous avons aussi montré la faisabilité d’utiliser des cellules fœtales dans le cadre du DPNI. Cinq cellules amniotiques microdisséquées, provenant de grossesses normales et aneuploïdes, ont suffi pour poser un diagnostic prénatal par une combinaison de l’amplification du génome complet et de la technique QF-PCR (réaction quantitative en fluorescence d’amplification entraînée par une polymérase) permettant la détection d’anomalies chromosomiques. Nos résultats ouvrent la voie à l’utilisation de cellules fœtales dans le sang maternel pour le DPNI.

A membrana amniótica humana vem assumindo um papel de extrema importância na medicina regenerativa nos últimos anos, principalmente na área de dermatologia e oftalmologia, sendo seu uso promissor no tratamento de doenças cuja terapêutica atual é pouco eficaz. Além disso, na membrana amniótica humana encontram-se células-tronco mesenquimais, que apresentam plasticidade e vem sendo aplicadas para a regeneração de tecidos. Entretanto, muito pouco se sabe sobre a capacidade plástica e o uso de células-tronco de membrana amniótica de animais, já que a literatura a este respeito, não é tão ampla quanto à de humanos. Neste projeto estabelecemos a cultura de células-tronco de membrana amniótica (MA) de fetos caninos, caracterizando as células in vitro e in vivo. As células de MA foram obtidas a partir de um procedimento cirúrgico de histerectomia em cadelas prenhas, durante campanhas de castração da prefeitura da cidade de São Paulo. As células de MA foram caracterizadas in vitro, observando-se características semelhantes a outras células-tronco mesenquimais. Porém, quando foi analisado o seu comportamento in vivo, observamos a formação de um tumor de crescimento rápido, aproximadamente um mês, após o inóculo dessas células em 10 camundongos imunossuprimidos nude, sendo o tumor identificado histologicamente como um carcinoma embrionário. Diante do comportamento biológico formando um tumor in vivo, inferimos que células-tronco provenientes de membrana amniótica de cães não devem ser usadas para aplicação com objetivos terapêuticos em animais, pelo menos até que novas coletas sejam realizadas para que se possa confirmar ou não este comportamento.
The human amniotic membrane has taken an extremely important role in regenerative medicine in recent years, especially in dermatology and ophthalmology, and its promising use in treating diseases for which current therapy is ineffective. In addition, the amniotic membrane has human mesenchymal stem cells, which exhibit plasticity and have been applied to tissue regeneration. However, very little is known about the plastic capacity and the use of stem cells from amniotic membrane of animals, since the literature in this regard, it is not as broad as those of humans. In this project we established a culture of amniotic stem cells of dog, characterizing these cells in vitro and in vivo. The cells were obtained from a surgical procedure for hysterectomy in pregnant bitches, during castration\'s campaigns of the São Paulo\'s municipality. The cells were characterized in vitro, observing characteristics similar to other mesenchymal stem cells. In spite of their behavior, in vivo we observed the formation of a fast-growing tumor about a month after the inoculation of these cells in 10 immunosuppressed mice, being the tumor identified histologically as an embryonic carcinoma. Considering the biological behavior of forming a tumor in vivo, we infer that stem cells from amniotic membrane of dogs should not be used for application for therapeutic purposes in animals, at least until other collections are carried out so that it can be confirmed or not.

A membrana amniótica é uma membrana translucida sendo a membrana mais interna da cavidade amniótica, formada por uma monocamada de células epiteliais disposta sobre uma membrana basal. Com o crescente interesse na utilização de células-tronco provenientes de anexos fetais, esta se torna uma promissora fonte de células-tronco. Sendo assim em trabalho anterior realizado pelo nosso grupo tivemos como objetivo, o estabelecimento da cultura celular e caracterização das células-tronco fetais de membrana amniótica de cão para verificar se a mesma pudesse ser uma nova fonte celular a ser usada nos protocolos de terapia celular, uma vez que os cães têm sido considerados modelos animais atraentes para avaliar novas drogas ou realizar ensaios pré-clínicos. As células de membrana amniótica obtidas a partir do trabalho anterior foram caracterizadas in vitro, observando-se características semelhantes a outras células-tronco mesenquimais. Porém, quando foi analisado o seu o seu potencial carcinogênico observamos a formação de um tumor de crescimento rápido, aproximadamente um mês, após o inóculo dessas células em 10 camundongos imunossuprimidos nude, sendo o tumor identificado histologicamente como um carcinoma embrionário. Diante deste comportamento acreditamos ser de extrema importância analisar células provenientes de novas coletas verificando se a mesmas podem se comportar como as anteriormente estudadas. Com isso, temos como objetivo deste trabalho estabelecer e caracterizar as células provenientes de duas novas coletas em diferentes períodos gestacionais visando verificar se estas células se comportam da mesma que as anteriores isso é se quando inoculadas nos animais formam tumor e assim poder ter certeza de que essas células são ou não, boas alternativas para terapia celular. As células obtidas nestas novas coletas têm características de células-tronco mesenquimais expressando alguns marcadores, tem curva de crescimento semelhante às células-tronco mesenquimais, se aderem ao plástico e se diferenciaram em adipócitos. Diferentemente das células obtidas no estudo anterior estas células não geraram tumor quando injetadas em camundongos imunossuprimidos, nude em até 60 dias após inoculação.
Amniotic membrane is a membrane translucent with the inner membrane of the amniotic cavity, formed by a monolayer of epithelial cells disposed on a basal membrane. With the growing interest in the use of stem cells from fetal membranes, this becomes a promising source of stem cells. So in a previous study conducted by our group we aim, the establishment of cell culture and characterization of fetal stem cells from amniotic membrane from dog to see if it could be a new source cell to be used in cell therapy protocols, Once the dogs have been considered attractive animal models to evaluate new drugs or performing pre-clinical tests. The cells from amniotic membrane obtained from previous work were characterized in vitro, observing characteristics similar to other mesenchymal stem cells. But when it was analyzed its its carcinogenic potential observed the formation of a fast-growing tumor, approximately one month after inoculation of these cells into immunocompromised nude mice 10, and the tumor identified histologically as embryonal carcinoma. Given this behavior we believe is extremely important to analyze cells from new collections by checking if the same can behave like those previously studied. With this, we aim of this work to establish and characterize the cells from two new collections at different gestational periods to verify whether these cells behave the same as the previous ones is that when inoculated into animals form tumor and thus be able to make sure that these cells are either not good alternatives for cell therapy. The cells obtained in these new collections has characteristics of mesenchymal stem cells expressing some markers, growth curve is similar to mesenchymal stem cells, adhere to plastic and differentiated into adipocytes. Unlike the cells obtained in the previous study these cells did not generate tumors when injected into immunocompromised mice, nude within 60 days after inoculation.

La drépanocytose est une maladie génétique héréditaire récessive causée par la substitution d'un acide aminé dans la chaîne β-globine aboutissant à la production d'hémoglobine anormale (HbS). Dans des conditions hypoxiques, l’HbS polymérise entraînant une falciformation et une perte de déformabilité des globules rouges (GR). Au cours de la drépanocytose, le dysfonctionnement splénique entraîne des complications potentiellement mortelles, en particulier chez les jeunes enfants. Généralement, une asplénie fonctionnelle précède la survenue d’une asplénie anatomique avec cependant une grande variabilité inter-individuelle du fait de facteurs modulateurs génétiques et environnementaux. L’hémoglobine fœtale (HbF) est le principal modulateur de la gravité de la maladie, car la présence d’HbF inhibe la polymérisation d’HbS, retardant et prévenant ainsi les complications graves, améliorant la qualité de vie des patients et augmentant leur survie. Il existe une hétérogénéité assez bien caractérisée de la concentration et de la distribution cellulaire d'HbF dans les GR circulants, mais le rôle de l'HbF au cours de l'érythropoïèse est mal documenté.Dans le but de mieux comprendre le rôle de l’HbF dans la perte de fonctionnalité splénique, la survie des GR et l'érythropoïèse inefficace, nous avons étudié 1) l'histoire naturelle du dysfonctionnement splénique chez les enfants drépanocytaires, 2) l'expression cellulaire et la distribution de l'HbF comparativement chez les enfants, chez les patients naïfs et ceux traités par hydroxycarbamide (HC) et 3) le rôle de l'HbF au cours de l'érythropoïèse terminale.Nous avons d’abord développé une méthode de cytométrie en flux à haut débit pour mesurer la fonction de filtration splénique et avons montré que la perte de fonction splénique est présente très tôt dans la vie, dès 3 à 6 mois chez les enfants drépanocytaires, puis diminue avec l'âge. Nous avons également mis en évidence que les cellules irréversiblement falciformées (ISC) jouaient un rôle dans la survenue de la séquestration splénique aiguë, laquelle entraîne à son tour une perte supplémentaire de fonction. Dans la deuxième partie de ce travail, nous avons mis en place une approche originale pour déterminer la distribution d'HbF à l’échelle cellulaire. En utilisant une cohorte longitudinale de patients traités par HC (inducteur d'HbF), nous avons montré que celui-ci avait un impact positif global sur les GR, en augmentant non seulement le contenu en HbF, mais également le volume de tous les GR, indépendamment de leur contenu en HbF. Nous avons par ailleurs montré que les cellules riches en HbF (High-F) étaient un marqueur précis d’efficacité de l'HC. Dans la dernière partie de la thèse, nous avons démontré pour la première fois une érythropoïèse inefficace chez les patients drépanocytaires et avons révélé un nouveau rôle de l'HbF au cours de l'érythropoïèse terminale, celui de protéger les érythroblastes de l'apoptose.En conclusion, ce travail montre que l'HbF a un effet bénéfique supplémentaire sur la drépanocytose en conférant non seulement une survie préférentielle des cellules F dans la circulation, mais en diminuant également l'érythropoïèse inefficace. Ce résultat suggère que la persistance d’HbF dans la drépanocytose serait davantage la conséquence d’un enrichissement en F-érythroblastes au cours de la différenciation érythroïde terminale survenant peu après la naissance plutôt qu’un retard du switch des hémoglobines
Sickle cell disease (SCD) is caused by a single point mutation in the β-globin gene generating sickle hemoglobin (HbS). Hypoxia drives HbS polymerization that is responsible for red blood cell (RBC) sickling and reduced deformability. In SCD, splenic dysfunction results in life-threatening complications, particularly in early childhood. During the course of the disease, the spleen functionally declines and anatomically disappears, although with great individual variability depending on modulating genetic and environmental factors. The key modulator of disease severity is fetal hemoglobin (HbF), as the presence of HbF inhibits HbS polymerization, thus delaying and preventing severe complications, ameliorating patients’ quality of life and increasing survival. There is a rather well characterized hetero cellular concentration of HbF and distribution in circulating RBCs but the role of HbF during erythropoiesis, is poorly documented. With the aim of better understanding the role of HbF in spleen function, red cell survival and ineffective erythropoiesis we investigated 1) the natural history of spleen dysfunction in SCD children, 2) the cellular expression and distribution of HbF in SCD children, in untreated patients and patients treated with Hydroxycarbamide and 3) ineffective erythropoiesis and the role of HbF during terminal erythropoiesis.We developed a flow cytometry high-throughput method to measure splenic filtration function and showed that splenic loss of function is present very early in life at 3-6 months in SCD children and further declines with age. We also highlighted that irreversibly sickled cells (ISCs) are a potential contributor to acute splenic sequestration (ASS) which in turn results in further loss of splenic function. In the second part of this work, we set up an original approach to determine HbF distribution per cell. Using a longitudinal cohort of patients treated with hydroxycarbamide (HC - an inducer of HbF), we showed that HC has a global positive impact on RBCs, by not only increasing HbF content but also by increasing the volume of all RBCs independent of HbF. We moreover showed that High F-cells are a more precise marker of HC efficacy. In the last part of the thesis, we showed for the first time clear evidence of ineffective erythropoiesis in SCD and revealed a new role of HbF during terminal erythropoiesis protecting erythroblasts from apoptosis. In conclusion, this work shows that HbF has an additional beneficial effect in SCD by not only conferring a preferential survival of F-cells in the circulation but also by decreasing ineffective erythropoiesis. Importantly, it suggests that the delay in hemoglobin switch in SCD might be also due to an enrichment in F-erythroblasts during terminal erythroid differentiation occurring very early in infancy, shortly after birth

Au cours des dernières décennies, nous avons progressivement vu augmenter un certain nombre d'anomalies de la fonction de reproduction masculine dans les pays industrialisés. Ces constatations ont fait émerger l'hypothèse selon laquelle certains polluants de notre environnement pourraient altérer le développement du testicule fœtal et ainsi être responsables de ces anomalies. Parmi les composants incriminés se trouvent les phtalates, largement répandus dans l'environnement. Ces composés ont été décrits comme reprotoxiques, ils altèrent le développement de la lignée germinale dans différentes espèces et entraînent une diminution de la production de testostérone chez le rat. Toutefois, très peu de données sont disponibles quant à leurs effets chez l'Homme. Dans cette étude, nous avons analysé les effets d'un phtalate, le MEHP, sur le développement du testicule fœtal humain au premier trimestre de la grossesse, dans un modèle de culture organotypique qui permet le maintien des différentes structures de l'organe. Nous avons tout d'abord démontré que le MEHP (10-4M) n'altère pas la production de testostérone du testicule fœtal humain, contrairement aux résultats décrits chez le rat. En revanche, nous avons montré que l'exposition au MEHP entraîne une rapide diminution du nombre de cellules germinales par apoptose. A la suite de ces résultats, nous avons testé l'effet de doses plus faibles de MEHP afin de se placer à des concentrations de phtalates ayant été mesurées dans les liquides biologiques. Nous avons ainsi démontré que les cellules germinales du testicule fœtal humain sont altérées suite à l'exposition à des doses de MEHP de 10-5M. Enfin, dans la 3ème partie de ce travail, nous nous sommes intéressés aux mécanismes d'action des phtalates. Différentes études, notamment dans le foie, démontrent l'implication des récepteurs nucléaires dans les effets de ces composés. Il nous a donc semblé important de rechercher leur implication dans les effets des phtalates sur le testicule fœtal. Nous avons démontré que LXRα est très certainement impliqué ces effets puisque l'expression des ARNm de ce récepteur est augmentée. Par ailleurs, ce récepteur nucléaire contrôle deux voies métaboliques, la synthèse de cholestérol et la synthèse des acides gras qui semblent toutes deux modulées par les phtalates dans le testicule fœtal humain. Enfin, nous avons montré que l'implication de ces voies métaboliques est commune entre la gonade mâle et la gonade femelle, sans pour autant que l'effet sur les cellules germinales mâles ai pu être mis en évidence dans l'ovaire fœtal. En conclusion, cette étude a contribué à caractériser les effets des phtalates sur la mise en place des fonctions de reproduction chez le fœtus humain. Nous avons également pu mettre en évidence un nouveau mécanisme de ces composés, impliquant la superfamille des récepteurs nucléaires ainsi que la synthèse du cholestérol et des acides gras.

INTRODUCTION: Intrauterine growth restriction (IUGR) due to placental insufficiency affects up to 7-10% of pregnancies and is a major cause of perinatal mortality and long term morbidity. IUGR results in low birth weight, which is associated with increased risk of cardiovascular mortality in adulthood, and is thought to be mediated by fetal cardiovascular programming. IUGR fetuses show signs of cardiac systolic and diastolic dysfunction from early stages, together with the appearance of biochemical signs of cell damage, changes in cardiac morphometry and function. These fetal changes persist permanently, resulting in vascular and cardiac remodelling. HYPOTHESIS: IUGR induces cardiac remodeling and dysfunction at organ, cellular and organelle level. OBJECTIVE: To characterize cardiac remodeling and function in IUGR, at organ, cellular and organelle level. METHODOLOGY: Cardiac remodeling due to IUGR has been evaluated in in two study groups: 1) human IUGR fetuses; 2) an experimental model of IUGR in New Zealand rabbit which has been previously validated. Several imaging techniques have been used to evaluate cardiac remodeling in IUGR: 1) X-ray synchrotron radiation to evaluate detailed cardiac anatomy; 2) transmission electron microscopy to study the intracellular organization of the cardiomyocyte; 3) multiphoton and second harmonic generation microscopy to evalaute the morphometry and ultrastructure of the sarcomere. Additionally, stuctural changes have been accompained with the evaluation of cardiac function with echocardiography and the evaluation of the gene expression profile. Cardiac remodeling has been evaluated in fetuses and the postnatal persistence of some of the changes has been assessed. RESULTS: At the organ level, fiber orientation is altered in IUGR fetal hearts and coronary vessels appear to be dilated. The intracellular organization of the cardiomyocyte of IUGR fetal hearts presents changes affecting the relationship between mitochondria and myofilaments. At the level of the sarcomere, which is the main contractile unit, sarcomere length results to be shorter in IUGR fetal hearts. The latest persists in postnatal life. Signs of cardiac remodeling of IUGR hearts are associated to a subclinical cardiac dysfunction and changes in the expression of groups of genes functionally related, involved in oxygen homeostasis, energy production and the M-band of the sarcomere. CONCLUSIONS: Signs of cardiac remodeling induced by IUGR are described in this Thesis, from the whole cardiac architecture, through the cardiomyocyte intracellular organization, to the ultrastructure of the sarcomere. Moreover, the structural changes are associated with cardiac dysfunction and gene expression alterations. Specifically, we demonstrate: 1) the detailed cardiac anatomy is different in IUGR fetal hearts, with changes in fiber orientation and coronary vessels dilation; 2) the arrangement of the intracellular organelles of the cardiomyoycte is altered in IUGR fetuses, affecting mitochondria and their interaction with myofilaments; 3) sarcomere length is shorter in IUGR fetuses, which could result in decreased contractility; 4) shorter sarcomere length persists in postnatal life, suggesting that IUGR-induced changes on sarcomere morphometry during fetal life could contribute to the increased risk of cardiovascular disease in adulthood; 5) the described changes of cardiac remodeling are associated with subclinical cardiac dysfunction in IUGR fetuses and changes in the expression of groups of genes involved in oxygen homeostasis, energy production and the sarcomere M¬band.
INTRODUCCIÓN: La restricción de crecimiento intrauterino (CIR) debido a insuficiencia placentaria afecta a un 7-10% de las gestaciones y constituye la mayor causa de mortalidad perinatal i morbilidad a largo plazo. El CIR resulta en bajo peso al nacer, lo que se relaciona con un incremento de la mortalidad por causas cardiovasculares en edad adulta, y se cree que está mediado por el concepto de “programación fetal” de la función cardiovascular. HIPÓTESIS: El CIR induce un remodelado cardiaco acompañado de disfunción cardiaca, a nivel del órgano, célula y orgánulo. OBJETIVOS: Caracterización del remodelado y función cardiaca en el CIR, a nivel de órgano, célula y orgánulo. METODOLOGÍA: Estudio del remodelado cardíaco en CIR en dos grupos de estudio: 1) fetos CIR humanos; 2) modelo animal de CIR previamente validado. Se han usado distintas técnicas avanzadas de imagen para estudiar el remodelado cardiaco en CIR: 1) micro-CT por rayos X generados por sincrotrón para el estudio de la arquitectura detallada del corazón; 2) microscopía electrónica de transmisión para la evaluación de la organización intracelular del cardiomiocito; 3) microscopia multifotón e imagen de segundo harmónico para el estudio de la morfometría del sarcómero. Se ha evaluado la función cardiaca mediante ecocardiografia y análisis bioinformático del perfil de expresión génica. RESULTADOS: A nivel de órgano, la orientación de las fibras se encuentra alterada en fetos CIR y los vasos coronarios dilatados. A nivel del cardiomicito, la organización intracelular presenta cambios afectando mayormente la relación entre mitocondrias y miofilamentos. La longitud del sarcómero, unidad contráctil fundamental, es inferior en corazones de fetos CIR. Esta última alteración persiste en vida postnatal. Los cambios estructurales de los corazones CIR se acompañan de signos de disfunción cardiaca subclínica y cambios en la expresión de grupos de genes funcionalmente relacionados, involucrados en la homeostasis del oxígeno, la producción de energía y la banda M del sarcómero. CONCLUSIONES: Los cambios descritos proporcionan evidencias de los mecanismos involucrados en el remodelado cardíaco asociado al CIR. Además, dichos cambios estructurales se asocian con una disfunción cardíaca y cambios a nivel de expresión génica.

Objetivo: Estudar os efeitos do emprego de dois materiais consideravelmente diferentes quanto à origem e custo na correção intra-uterina da meningomielocele criada experimentalmente em feto de ovino. Métodos: Em 36 fetos de ovinos foi criado um defeito aberto de tubo neural, com 75 de dias de gestação. Os casos foram divididos em três grupos: o controle onde o defeito não foi corrigido, grupo corrigido A onde o material utilizado para cobrir a medula exposta foi a matriz dérmica humana acelular (MDHA) e o grupo corrigido B onde o material foi a celulose biossintética (CB). Após a correção realizada com 100 dias, os fetos eram mantidos intra-útero até o termo da gestação. Os sacrifícios foram realizados com 140 dias e a coluna fetal era submetida à análise macro e microscópica onde foi observada a aderência dos materiais à pele, medula ou tecido nervoso remanescente. Resultados: Na fase inicial (piloto), 11 fetos foram operados e 4 sobreviveram (37%). Na segunda fase (estudo) 25 fetos foram operados e 17 sobreviveram (68%). No grupo de estudo, 6 fetos não foram submetidos à correção (grupo controle), 11 casos foram corrigidos e ocorreu 1 perda fetal. Do total de 10 casos, 4 constituíram o grupo A e 6, o grupo B. À macroscopia observou-se deslizamento da pele e tecidos subjacentes sobre a CB em todos os casos onde ela foi empregada e isto não ocorreu em nenhum dos casos onde a MDHA foi utilizada. Para comparar a aderência, foram considerados 4 casos do grupo A e 4 do grupo B. A aderência, caracterizada pela migração de células do hospedeiro e proliferação de vasos para dentro da MDHA, foi observada em 100% dos casos do grupo A e em nenhum caso no grupo B (p < 0,05). No grupo B observou-se formação de uma camada de fibroblastos ao redor do material, protegendo a medula, caracterizando a formação de uma \"neoduramater\". Conclusão: A utilização da película de celulose biossintética parece ser mais adequada como substituto de dura-máter para cobertura e proteção do tecido nervoso que a matriz dérmica humana acelular. Ela parece promissora na correção intra-uterina da meningomielocele, evitando a aderência do tecido nervoso aos planos superficiais (\"medula presa\") minimizando os efeitos deletérios do ambiente intra-uterino sobre a medula espinhal.
Purpose: The aim of this study was to compare the effectiveness of two dura-mater substitutes, namely human acellular dermal matrix (HADM) and biosynthetic cellulose (BC), in repairing, in utero, surgically-induced meningomyelocele (MMC) in fetal sheep. Methods: A neural tube defect was created at 74-77 days gestation in 36 fetal sheep. They were divided into 3 groups, the control group that did not receive pre-natal corrective surgery, and the other two groups that received corrective surgery using HADM (Group A) or BC (Group B). Both materials were used as a dura-mater substitutes between the neural tissue and the sutured skin. Correction was performed at gestation day 100 and the fetuses were maintained in utero until term. Sheep were sacrificed on gestation day 140. The fetal spine was submitted to macro and microscopic analysis. At microscopy, adherence of the material to the skin and neural tissue was analyzed. Results: In the initial phase (pilot), experimentally-induced MMC was performed on 11 fetuses and 4 survived (37%). In the second phase (study), 25 fetuses received surgery and 17 survived (68%). In the study group, 6 fetuses did not undergo repair (control group), 11 cases were submitted to corrective surgery (experimental group) and one fetal loss occurred. Of the surviving cases in the experimental group, 4 constituted Group A and 6 in Group B. Macroscopically, skin and underlying tissues where easily displaced from the BC in all cases it was used; in contrast, HADM adhered to these tissues. To compare the adherence, 4 cases from Group A and 4 in Group B were studied. We observed adherence, host cell migration and vessel proliferation into the HADM all sections from Group A and this aspect was not present in any cases in Group B (p < 0.05). In Group B, we also observed that a new fibroblast layer formed around the BC thus protecting the medulla and constituting a \"neoduramater\". Conclusion: The use of BC seems to be more adequate as a dura-mater substitute to cover the damaged neural tissue than HADM. It seems promising for use in the in utero correction of MMC because to does not adhere to neural tissue of superficial and deep layers (\"tethered spinal cord\"). Thus, BC minimizes the mechanical and chemical intrauterine damage to the spinal medulla.

GNAS est un locus complexe soumis à l'empreinte parentale. Il code pour cinq transcrits alternatifs dont l’expression est régulée de manière parentale, tissulaire et développementale : la sous-Unité alpha stimulatrice de la protéine G hétérotrimérique (Gαs), XLαs, NESP55, et deux ARNnc, A/B et GNAS-AS1. Gαs est une protéine clé dans la transduction hormonale partageant avec XLαs la capacité de produire l'AMPc intracellulaire après stimulation des récepteurs couplés à Gαs.Dans la première partie de ma thèse, je me suis concentrée sur l'étude du rôle des transcrits de GNAS, en particulier XLαs, dans la croissance fœtale et post-Natale. J’ai profité du modèle unique des pseudohypoparathyroïdies (PHPs), pathologies humaines rare de l’empreinte, causées par des anomalies génétiques ou épigénétiques du locus GNAS altérant le dosage génique des transcrits de GNAS. La croissance anormale est une caractéristique majeure des PHPs.Dans la deuxième partie de ma thèse, j’ai étudié le profil épigénétique du locus GNAS (méthylation de l'ADN et expression des transcrits) dans les cellules souches humaines embryonnaires -HESCs-, dans les cellules pluripotentes induites dérivées à partir de fibroblastes de sujets sains -IPSCs- et dans les cellules redifférenciées en cellules souches neurales et mésenchymateuses. La caractérisation précise du locus humain GNAS en physiologie (cellules souches) et pathologie (PHP) est essentielle pour une meilleure compréhension des processus développementaux importants comme la croissance. L'exploration du phénotype "croissance" de différents types de PHPs a permis de mieux comprendre le rôle des transcrits du locus GNAS dans la physiologie et la physiopathologie. L'analyse de cellules des PHPs a permis de mieux caractériser l’impact des anomalies moléculaires du locus GNAS en pathologie humaine. Les hiPSCs peuvent être un outil utile pour étudier les modifications épigénétiques au niveau du locus GNAS
GNAS is a complex locus subjected to parental imprinting encoding five parental-, tissue- and developmental-Manner regulated transcripts : the alpha stimulatory subunit of the G protein (Gαs), XLαs, NESP55, and two ncRNAs, A/B and the antisens GNAS-AS1. Gαs is a key protein in hormonal signaling sharing with XLαs the ability to produce intracellular cAMP upon stimulation of Gαs-Coupled receptors. In the first part of my thesis, I focused on studying the role of the GNAS transcripts, particularly XLαs, in fetal and postnatal growth. I took advantage of the unique model of pseudohypoparathyroidism (PHP), a rare human disease, caused by genetic or epigenetic abnormalities at the GNAS locus leading to various combinations of GNAS transcripts alterations. Abnormal growth appears to be a major feature of PHP. In the second part of my thesis, I studied the epigenetic pattern of GNAS (DNA methylation and transcripts expression) in human embryonic stem cells -HESCs-, in induced pluripotent stem cells -IPSCs- derived from fibroblasts from healthy individuals, and in cells re-Differentiated from these stem cells in neuronal and mesenchymal cells. The precise characterization of the human GNAS locus in physiology (stem cells) and pathology (PHP) is critical for a better understanding of major processes like growth. Through exploration of the "growth" phenotype of different groups of PHPs we have participated to the better understanding of the role of the GNAS transcripts in the physiology and pathophysiology. Human iPSCs may be an useful tool to study epigenetic modifications at the GNAS locus

L’activation des lymphocytes T nécessite un double signal. Le premier est antigénique et permet la reconnaissance d’un peptide spécifique présenté à la surface de cellules présentatrices d’antigène (APC). Le second signal est co-stimulateur et implique l’interaction avec des molécules activatrices exprimées par les APC et la présence de cytokines proinflammatoires. Les cellules dendritiques (DC) sont les uniques APC capables de délivrer ce double signal et d'activer les lymphocytes T naïfs, initiant ainsi les réponses immunes primaires. L’immaturité du système immunitaire du nouveau-né est responsable d’une plus grande susceptibilité aux maladies infectieuses ainsi qu’une faible réponse vaccinale. Des déficiences tant au niveau de l’immunité innée que de l’immunité acquise participe à une faible défense face aux agressions. A la naissance, les DC expriment des niveaux faibles de molécules co stimulatrices et présentent un défaut majeur de synthèse d'IL 12, cytokine cruciale pour l’établissement de réponses de type Th1. Le but de ce travail est d’évaluer la capacité des DC du nouveau-né humain à activer les lymphocytes T CD8+.

Dans une première approche, nous avons utilisé un modèle unique d’induction de réponse primaire in vitro qui permet d'étudier l'activation de lymphocytes T CD8+ spécifiques de l’antigène Melan-A, une protéine du soi exprimée par les mélanocytes. Ces lymphocytes existent à des fréquences particulièrement élevées chez les individus sains HLA-A2 et présentent les caractéristiques de lymphocytes T naïfs. Dans ce modèle, nous avons d’abord analysé les capacités immunostimulatrices de différentes populations de DC différenciées in vitro. Nous avons observé que les DC différenciées par la culture de monocytes purifiés en présence d'IL-3 et d’IFN-beta sont capables d’initier une réponse fonctionnelle des lymphocytes T CD8+, analogue à celle induite par les DC différenciées en présence de GM-CSF et d’IL-4. Ce même modèle nous a permis de démontrer que, en dépit de leur défaut de production d’IL 12, les DC du nouveau-né sont capables d'induire efficacement une réponse lymphocytaire T CD8+ cytotoxique.

Afin dévaluer la relevance in vivo de nos observations, nous avons étudié le phénotype et la fonction des DC circulantes chez des nouveau-nés infectés par le cytomégalovirus (CMV). L’infection par le CMV au cours de la vie fœtale représente une situation clinique où le nouveau-né développe une réponse mature et fonctionnelle des lymphocytes T CD8+, alors que celle des lymphocytes T CD4+ est déficiente. Ces expériences ont montré que le phénotype, la fonction et la réponse à différents stimuli des APC présentes en périphérie ne sont pas affectés par l’infection congénitale par le CMV. Ces résultats suggèrent que l’observation des DC circulantes des nouveau-nés infectés par le CMV ne permet pas d’analyser l’influence du virus sur la fonction des DC néonatales. Dans ce but, nous avons reproduit un modèle d’infection in vitro de DC par une souche primaire du CMV. L’utilisation de micropuces à ADN nous a permis de comparer l’expression de gènes différentiellement induits par l’infection des DC d’adultes et de nouveau-nés. Nous avons ainsi révélé une proportion importante de gènes différentiellement induits, parmi lesquels celui de l’IFN-beta. Nous avons confirmé ce défaut au niveau protéique et mis en évidence une production d’IL 12 déficiente en réponse à l’infection par CMV.

L’ensemble de nos résultats indique que malgré leur immaturité, les DC du nouveau-né sont capables, dans certaines circonstances, d’induire une réponse lymphocytaire T CD8+ cytotoxique. Cependant, le défaut de production de certaines cytokines co-stimulatrices pourrait être impliqué dans la faible réponse des lymphocytes T CD4+ à l’infection par CMV. Ces observations ont d’importantes implications pour la compréhension de l’induction de réponses cytotoxiques au cours d’infections virales et pour l’élaboration de stratégies vaccinales en début de vie.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

El mielomeningocele (MMC), la forma más severa de espina bífida, es una malformación del tubo neural que se presenta en tres de cada diez mil nacimientos, y que provoca un grave daño neurológico y un complejo cuadro clínico en el feto en desarrollo. Aunque sus causas son desconocidas, se está ahondando en el conocimiento de las mismas así como en el desarrollo de nuevas terapias, incluyendo las basadas en medicina regenerativa que se dirigen a la reparación prenatal del defecto, mediante estrategias de regeneración del tejido neural dañado con el fin de revertir el daño causado por el defecto congénito sobre la médula espinal. En este estudio, se analizaron los líquidos amnióticos (LA) de fetos sanos y afectos con MMC para caracterizar los distintos tipos celulares e identificar y aislar las células con propiedades de precursores neurales; también se analizaron las diferentes poblaciones celulares presentes en el líquido cefalorraquídeo (LCF) procedentes de muestras de pacientes afectados por MMC y se evaluó su capacidad de diferenciación in vitro a células de linaje neural con el fin de demostrar sus propiedades como progenitores neurales (NPCs). Se ha demostrado la presencia de NPCs en el LA de pacientes con MMC que no se observaron en muestras de LA de individuos sanos. Se pudo demostrar que estas células se encuentran presentes en el LCF y que su presencia en el LA de fetos con MMC se debe posiblemente a la pérdida de LCF desde el canal medular hacia la cavidad amniótica a través del defecto de MMC, por lo que pueden ser idóneas para una futura estrategia terapéutica basada en la terapia celular en pacientes con MMC. Una vez demostrado el potencial de estas células como progenitores neurales, se llevaron a cabo ensayos de trasplante celular en modelos animales. En primer lugar se ensayó la técnica de trasplante celular en un nuevo modelo de MMC quirúrgico en conejo y tras demostrar que estas células permanecen en el lugar del trasplante y que no se vio afectada su viabilidad tras la implantación sobre la zona lesionada de la médula espinal, se extrapoló esta metodología al modelo de MMC en oveja, consiguiendo también resultados positivos, y demostrando la presencia y viabilidad de las células trasplantadas más de dos meses después de la realización del trasplante y sin haberse producido rechazo inmunológico. Los prometedores resultados obtenidos en el presente trabajo podrían ser la antesala para el desarrollo de una novedosa estrategia basada en terapia celular para la regeneración del tejido neural de la médula espinal mediante intervención prenatal en fetos con MMC. Esta nueva estrategia terapéutica podría permitir el ensayo de la terapia celular basada en el trasplante autólogo de NPCs aisladas a partir del LA del propio paciente en futuros ensayos clínicos en pacientes con espina bífida.
Myelomeningocele (MMC), the most severe type of spina bifida, affects approximately 3.3 in 10,000 live births and represents one of the most debilitating birth defects in humans, affecting severely the spinal cord and brain during gestation. MMC results in variable disabilities including paraplegia, hydrocephalus, sexual dysfunction, skeletal deformations and impaired mental development. Defects on primary neurulation could leads to failure of neural tube closure and MMC creation; so, unprotected spinal cord is subjected to progressive damage with advancing gestational age, probably due to chemical and mechanical factors related to exposure to the intrauterine environment and the continuous loss of cerebrospinal fluid (CSF) through the defect, causing a hindbrain herniation or Chiari II malformation and hydrocephalus. It is hypothesized that MMC could be induced by “two hits”; the first due to failure of neural tube closure and the second, the degeneration of neural tissues due to the exposition to the utero environment. Midgestational fetal coverage of spinal defect has demonstrated to minimize damage to developing central nervous system, but, this technique only prevent additional damage so the previous neural injury persist after birth. Previous studies have stated the presence of neural progenitor cells (NPCs) in MMC amniotic fluid (AF) samples, hypothetically, these NPCs come from CSF. In the present study we isolated NPCs from AF and CSF samples, evaluated the NPCs growing and differentiation capacities and we transplanted NPCs in a newly developed surgical-chemical MMC model in fetal rabbits by the implantation of NPCs embedded in an adequate scaffold in the site of spinal cord injury. Then we made a step forward by transferring this methodology to the surgically-induced MMC model in fetal sheep. The creation of the injury was at 75 day of gestation and the repair of MMC with NPCs isolated from CSF was performed at 95 days of gestation, demonstrating a successful NPCs implantation in the injured spinal cord more than 2 months later. These promising results could be paving the way to the development of new cell therapy- based approaches to regenerate neural tissues, based on autologous NPCs transplantation during the prenatal surgery intervention in MMC patients.

A Asma vem apresentando taxas de prevalência crescentes em todo o mundo. Os objetivos deste trabalho são avaliar a presença de elementos celulares e humorais indicativos em sangue e colostro de mães asmáticas e saudáveis e no sangue de cordão umbilical de seus recém-nascidos (RN). Observamos menor produção de IgG pelas mães asmáticas. Células dendríticas de mães asmáticas possuem maior expressão de CD80 e CD86. Mães asmáticas possuem mais células de memória central. Linfócitos T CD4+ de mães asmáticas produzem níveis maiores IFN-g. Células CD3+ e CD4+ de mães asmáticas produziram mais IL-13. Mães saudáveis produziram maiores quantidades de IL-10. Concluímos que mães asmáticas possuem menores níveis de IgG e IgM, o que parece aumentar dos níveis de IgE, mães asmáticas possuem mais células de memória central, linfócitos T CD4+ produzem maiores quantidades de citocinas como IL-13 e IFN-g, células dendríticas de mães asmáticas possuem maior expressão das moléculas co-estimulatórias, assim como seus RNs possuem maior expressão de CD80. Células de mães asmáticas produzem níveis menores de IL-10, o colostro de mães atópicas não possui diferenças entre os parâmetros aqui estudados. O aleitamento materno deve ser indicado para mães asmáticas e seus filhos.
Asthma has been presenting higher prevalence rates worldwide. The objective of this work is to evaluate the presence of cellular elements and humoral indicative in blood and colostrums of healthy and asthmatic mothers and in cord umbilical blood of their newborn (NB). Lower production of IgG from asthmatic mothers was observed. Dendritic cells of asthmatic mothers have higher CD80 and CD86 expression. Asthmatic mothers have more central memory cells. TCD4+ lymphocyte of asthmatic mothers produce higher levels of IFN-g. CD3+ and CD4+ cells of asthmatic mothers produce higher quantity of IL-13. Healthy mothers produce higher quantity of IL-10. We conclude that asthmatic mothers have lower levels of IgG and IgM, and this seems to raise the IgE levels, asthmatic mothers have more central memory cells, CD4+ T lymphocyte and produce higher quantities of cytokines such as Il-13 and IFN-g. Dendritic cells of asthmatic mothers have higher expression of costimulatory molecules, as well as their NBs have higher expression CD80. Cells of asthmatic mothers produce lower levels of IL-10, the colostrums of atopic mothers does not have differences in the parameters here studied. The maternal breastfeeding must be indicated for asthmatic mothers and their of spring.

L’objectif principal de ce projet de recherche doctorale est la mise au point d’approches méthodologiques fiables et reproductibles permettant la caractérisation génétique de cellules rares circulantes isolées par la méthode de filtration ISET® (Rarecells®, France). La première application développée consiste en la détection des mutations du gène suppresseur de tumeur VHL (Von Hippel Lindau) dans les cellules rares circulantes (CRC) uniques isolées du sang de 30 patients atteints de carcinome rénal à cellules claires (CRCC), et réalisée comparativement à l’analyse cytopathologique. L’étude génétique a également été conduite en parallèle dans les 30 tissus tumoraux correspondants. Les résultats ont mis en lumière une potentielle complémentarité de l’approche de génétique moléculaire sur cellules uniques avec l’analyse cytomorphologique de référence et suggèrent que combiner ces approches permettrait d’obtenir une plus grande sensibilité de détection des cellules cancéreuses circulantes chez les patients atteints de CRCC. Une deuxième application a consisté en le développement d’une approche innovante pour le diagnostic prénatal non-invasif des maladies génétiques récessives par analyse de cellules trophoblastiques rares collectées au niveau du col de l’utérus. Enfin, des développements supplémentaires ont permis d’optimiser les analyses de séquençage à haut débit et de les appliquer à des CRC individuelles isolées par ISET®. Cette nouvelle approche, associée à l’isolement de CRC non fixées, est en mesure de fournir des données génétiques élargies à l’exome entier pour des applications à la fois en oncologie prédictive et en diagnostic prénatal non invasif
The aim of this doctoral research project is the development of reliable and reproducible methodological approaches enabling the genetic characterization of circulating rare cells (CRC) isolated by ISET® filtration (Rarecells®, France). The first application developed consists in detecting mutations of the VHL (Von Hippel Lindau) tumor suppressor gene in single CRC isolated from the blood of 30 patients patients with clear cell renal cell carcinoma (ccRCC), assessed according to the results obtained by cytopathological analysis. In parallel, genetic analysis of VHL mutations was conducted in the corresponding tumor tissues. Results revealed a potential complementarity of the molecular genetic approach targeted to single cells with the reference method of cytopathological analysis and suggested that combining both strategies could improve the sensitivity of circulating cancer cells’ detection in patients with ccRCC. A second application consisted in the development of an innovative approach for non-invasive prenatal diagnosis of recessive genetic diseases by analysis of rare trophoblastic cells collected from the cervix. Finally, further developments allowed to optimize high-throughput sequencing analyses and to apply them to single CRC isolated by ISET®. This approach, combined with the isolation of living CRC, enabled us to obtain broader genetic data from the whole exome and should foster innovative applications to both predictive oncology and non-invasive prenatal diagnosis

L’inflammation joue un rôle central dans la rupture des membranes fœtales (MF), que celle-ci ait lieu à terme ou prématurément, mais l’ensemble des mécanismes reste encore à élucider. Dans ce contexte, les études sur les inflammasomes, un des acteurs clés de l’inflammation, se sont récemment intensifiées. Ces plateformes intracellulaires, formées suite à un signal pro-inflammatoire, sont impliquées dans la mise en place et la propagation d’une réaction inflammatoire. Leur fonction au sein des MF commence à être décrite mais de nombreuses zones d’ombre persistent. L’objectif de ce travail a donc été de compléter la caractérisation des processus inflammatoires dépendant des inflammasomes dans les MF, en se focalisant sur les inflammasomes de type NLRP.Les inflammasomes NLRP sont constitués d’un récepteur NLRP, de l’adaptateur ASC et de la pro-caspase-1. Après avoir vérifié la présence de ces acteurs dans les MF à terme, un intérêt particulier a été porté à l’inflammasome de type NLRP7. En effet, sa fonction a déjà été étudiée dans la sphère placentaire mais jamais dans les MF. La stimulation de cellules épithéliales amniocytaires primaires avec un ligand spécifique de l’inflammasome NLRP7 a permis de montrer (i) l’augmentation du niveau protéique des trois acteurs de cet inflammasome (NLRP7, ASC et pro-caspase-1), (ii) la formation de l’inflammasome par co-localisation entre NLRP7 et ASC, (iii) l’activation de cet inflammasome montré par le clivage de deux effecteurs terminaux, la pro-caspase-1 et la gasdermine D. Ces résultats indiquent pour la première fois que les MF sont capables de mettre en jeu la signalisation de l’inflammasome NLRP7 en réponse à un signal pro-inflammatoire.En parallèle, deux activateurs naturels de l’inflammasome NLRP7 ont été identifiés pour la première fois dans les MF humaines à terme : il s’agit de Mycoplasma salivarium et Mycoplasma fermentans. Leur présence suggère le fait que l’inflammasome NLRP7 puisse jouer un rôle majeur dans les processus inflammatoires au sein des MF. L’ensemble de ce travail suggère donc fortement l’implication de l’inflammasome NLRP7 dans la physiopathologie de la rupture des membranes fœtales humaines, qui pourrait être une cible thérapeutique potentielle pour prévenir les ruptures prématurées des membranes fœtales
Inflammation plays a pivotal role in term or preterm fetal membranes (FM) rupture, but the detailed mechanisms remain unclear. In this context, studies on inflammasomes, one of the key inflammation actors, recently intensified. These intracellular platforms, formed following a pro-inflammatory signal, are involved in the establishment and propagation of an inflammatory reaction. Their functions in FM begin to be described but grey areas remain. Thus, the aim of this work was to complete the characterization of inflammasomes-dependent inflammatory processes, focusing on NLRP inflammasomes.NLRP inflammasomes are composed of a NLRP receptor, the adapter ASC and the pro-caspase-1. After verifying the presence of these actors in term human FM, we focused our interest on NLRP7 inflammasome. Indeed, its function has been studied in the placental area but never in FM. The stimulation of primary amnion epithelial cells with an NLRP7 inflammasome specific ligand demonstrated (i) an increased protein level of the three actors of this inflammasome (NLRP7, ASC and pro-caspase-1), (ii) the formation of this inflammasome by NLRP7 and ASC colocalization and (iii) the activation of this inflammasome, by cleavages of two end-effectors, pro-caspase-1 and gasdermin D. These results indicate for the first time that FM are able to activate NLRP7 inflammasome signalization in response to a pro-inflammatory signal. Moreover, two natural activators of NLRP7 inflammasome have been newly identified in term human FM: Mycoplasma salivarium and Mycoplasma fermentans. Their presence suggests that NLRP7 inflammasome could play an essential role in inflammatory processes in FM. All this work strongly suggests the involvement of NLRP7 inflammasome in pathophysiology of human FM rupture, which could be a potential therapeutic target to prevent premature rupture of FM

La mise en place de la lignée germinale au cours du développement constitue une des étapes fondamentales conditionnant la fertilité de l’individu adulte. Au cours des dernières décennies, le nombre croissant de couples consultant pour une aide médicale à la procréation a fait émerger l’hypothèse d’une altération des fonctions de reproduction chez l’Homme qui pourrait trouver son origine dans la perturbation du développement précoce. Dans l’ovaire fœtal, les cellules germinales s’orienteront vers la voie de l’ovogénèse, caractérisée entre autres par l’entrée en méiose de ces cellules. La majorité des données actuelles relatives à ces évènements sont issues du modèle murin alors que le développement de l’ovaire humain est significativement diffèrent de celui de la souris. Il est donc nécessaire d’approfondir nos connaissances du développement ovarien humain et d’identifier ses éventuelles perturbations. L’objectif de mon travail a été de mettre au point un outil d’étude du développement ovarien et d’identifier de nouvelles voies impliquées dans la régulation de l’entrée en méiose des cellules germinales fœtales humaines et leurs perturbations éventuelles.Nous avons mis au point un nouveau modèle de xénogreffe d’ovaires fœtaux humains du premier trimestre de gestation (au moment de l’apparition des premières cellules méiotiques). Ce modèle nous a permis d’observer un développement de l’organe et une différenciation des cellules germinales similaires à ceux observés in vivo. Ce modèle permettra des travaux à des âges auxquels le matériel d’étude est peu accessible. En couplant ce modèle de xénogreffe à une stratégie d’ARN-interférence, il nous a été possible d’inhiber l’expression d’un gène spécifiquement exprimé dans les cellules germinales ovariennes, DMRTA2, et de mettre en évidence un potentiel rôle de ce gène dans leur différenciation pré-méiotique. Nous avons observé une diminution du nombre de cellules ayant initié la méiose après inhibition de l’expression de ce gène. Par ailleurs, nous avons également identifié la présence dans l’ovaire fœtal de nombreux marqueurs décrits comme testiculaires chez la souris (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). L’expression de ces marqueurs pourrait expliquer la présence de cellules mitotiques tardives dans l’ovaire fœtal humain que nous avons pu observer jusqu’à 30 semaines de gestation. En parallèle de ces travaux, nous avons testé la sensibilité des cellules germinales à la dexaméthasone, glucocorticoïde pouvant être administré au cours de la grossesse. Il a été observé une augmentation de l’expression de PLZF, gène cible de l’activation des récepteurs aux glucocorticoïdes, pouvant expliquer la diminution du nombre de cellules germinales.En conclusion, ce travail de thèse a permis d’identifier un nouveau gène potentiellement régulateur de la transition mitose/méiose dans l’ovaire humain, et d’affiner nos connaissances sur le développement de l’ovaire humain et l’entrée en méiose des cellules germinales. Toutefois, de nombreuses questions restent posées ainsi de futures études devront clarifier si les cellules germinales mitotiques observées à des stades tardifs sont capables de se différencier en ovocytes compétents
Woman fertility is partially dictated by the set up of the human female germ line. During the last ten years, which saw an increased number of couples consulting for assisted reproductive cares, the hypothesis of an early alteration in reproduction functions has emerged.In the fetal ovary, germ cells enter the path of oogenesis differentiation characterized by meiotic initiation. On this subject, vast majority of the scientific data are obtained from the mouse model, even if differences with human ovarian physiology are widely acknowledged. Therefore it is necessary to extend our knowledge on human ovarian development and identify its perturbations. The objective of my work was to assess a new model to study ovarian growth, studying regulation of meiotic entry and perturbation of germ line differentiation.We sat up a new xenograft model of early human fetal ovaries, when very early meiotic germ cells appear. Organ growth and germ cells differentiation were comparable with in vivo observations. Using this model with an RNA-interference strategy, we inhibited the expression of an oogonia germ cell gene, DMRTA2. This inhibition conducted to a significantly reduced number of germ cells gene that initiated meiosis and DMRTA2 seemed to be required for mitotic-meiotic transition. In another hand, we identified, in the ovary, the expression of germ cells markers described as specifically male in rodent (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). The expression of these markers in the human ovary could explain the observation of mitotic germ cells in late fetal ovaries (30 wpf).In parallel, we tested germ cells sensibility to a synthetic glucocorticoid, dexamethasone, administrated during pregnancy in some justified pathologies. We observed an increased expression of PLZF that could explain the decreased number of germ cells observed in treated ovaries.In conclusion, we identified a new gene expressed in human fetal ovaries, potentially involved in the meiotic entry, and we extended our knowledge to characterized human germ line development. However, many points have to be clarified, as the possible competence of late mitotic germ cells to form oocytes

Le développement de stratégies thérapeutiques curatives pour les patients affectés de β-hémoglobinopathies, c’est à dire drépanocytose et β-thalassémies, fait face à une grande demande. En effet, un accès restreint à des donneurs compatibles limite l’unique thérapie définitive autorisée, la transplantation allogénique de cellules souches hématopoïétiques (CSH). Au cours de la première partie de cette thèse,nous avons optimisé une alternative thérapeutique,la transplantation autoloque de CSH corrigées par le biais de lentivirus (LV). Le développement de lentivirus exprimant la β-globine et l’amélioration des conditions de transduction des CSH a débouché sur de probants bénéfices cliniques pour les patients drépanocytaires et thalassémiques traités par cette approche dans le cadre d’essais cliniques récents. Malgré ces progrès majeurs,cette thérapie pourrait bénéficier d’améliorations. En effet,la correction de thalassémies majeures, dépendantes de transfusion, ainsi que de la drépanocytose requiert de forts niveaux d’expression du transgène. Le but de ce projet est de choisir un LV à titre élevé, capable de transduire une large portion de CSH et d’entraîner une forte expression du transgène dans des globules rouges (GR) dérivés de CSH. A cet effet, nous avons comparé différentes combinaisons d’éléments régulateurs afin de définir la cassette régulatrice minimale nécessaire pour l’obtention de forts niveaux d’expression de la β-globine. Nous avons construit 2 mini-LCRs contenant soit HS2 et HS3 (taille totale 2.6 kb) ou HS2, HS3 et HS4 (taille totale 3.7 kb) provenant du Locus Control Region (LCR). Ces cassettes ont été insérées dans les LV β-AS3 et β-AS3 HS4, respectivement, qui contrôlent l’expression d’une globine anti-falciformante, βAS3-globine.Premièrement, nous avons comparativement évalué l’efficacité de transduction des vecteurs β-AS3 et β-AS3 HS4 dans des cellules souches progéniteurs hématopoïétiques (CSPH) drépanocytaires et dans des CSH drépanocytaires. Le deuxième volet de cette étude a permis de déterminer l’expression de la βAS3-globine issue de ces deux constructions dans des GR dérivés de CSPH drépanocytaires, et d’évaluer l’efficacité du LV le plus efficace pour secourir le phénotype drépanocytaire.La seconde partie de cette thèse vise à développer une nouvelle stratégie thérapeutique par édition du génome pour réactiver l’hémoglobine fœtale (HbF). Cette approche s’appuie sur l’observation d’une meilleure évolution clinique des β-thalassémies et de la drépanocytose en présence de niveaux élevés d’HbF.Par l’utilisation de la technologie CRISPR/Cas9, nous avons invalidé les sites de liaisons de répresseurs transcriptionnels au niveau des promoteurs de la γ-globine pour réactiver l’expression de l’HbF dans des GR dérivés de CSPH drépanocytaires. La réactivation des gènes de γ-globine fœtale dans leur locus génomique endogène permet de s’affranchir des difficultés liées à l’expression relativement faible de transgène par copie de LV, principalement lié à la faible capacité des LV qui ne permet d’insérer que de courts fragments d’ADN du LCR, arrangés de manière non-physiologique. De plus,cette stratégie offre une approche potentiellement plus sûre comparée à une stratégie addition de gène basée sur l’usage de LV. Dans la drépanocytose, cette approche thérapeutique favorise l’expression de la γ-globine anti-falciformante aux dépens de la globine βS-globine mutée. Dans le cadre d’une approche comparative,nous avons évalué des cibles thérapeutiques connues et nouvelles au niveau des promoteurs de la γ-globine. A cet effet, plusieurs ARN guides (ARNg) ont été designés pour cibler 3 régions des promoteurs de la γ-globine, où des variants associés à des niveaux élevés d’HbF et/ou à des sites de liaison de répresseurs de l’HbF ont été décrits
Highly efficient curative therapeutic strategies are in great demand for patients affected by β-hemoglobinopathies, namely sickle cell disease (SCD) and β-thalassemia. Indeed, the poor access to compatible donors restrains the application of the only approved definitive therapy, the allogeneic hematopoietic stem cell (HSC) transplantation. In the first part of this thesis, we aimed at optimizing an established therapeutic alternative consisting in the autologous transplantation of lentivirus (LV)-corrected hematopoietic HSCs. The development of β-globin expressing LVs and the improvement of HSC transduction conditions have led to a clear clinical benefit for SCD and β-thalassemia patients treated with this approach in the frame of recent clinical trials. Despite these significant progresses, there is room for further improvement. Indeed, the correction of severe transfusion-dependent B-thalassemia and SCD patients requires high levels of transgene expression. The goal of this project was to select a high-titer LV able to transduce efficiently HSCs and to drive high levels of transgene expression in HSC-derived RBCs. To this purpose, we compared different combinations of regulatory elements, in order to define the minimal regulatory cassette needed for achieving high levels of globin expression in the frame of LVs. We constructed 2 mini-LCRs containing either HS2 and HS3 (total size 2.6 kb) or HS2, HS3 and HS4 (total size 3.7 kb) derived from the 16-kb Locus Control Region. These cassettes were inserted in the β-AS3 and β-AS3 HS4 LVs, respectively, driving the expression of an anti-sickling βAS3-globin transgene. First, we aimed at comparatively evaluate the transduction efficiency of β-AS3 and β-AS3 HS4 in SCD hematopoietic stem progenitor cells (HSPCs) and long term-repopulating HSCs. The second aim of the study was to assess β-AS3 and β-AS3 HS4 derived transgene expression in RBCs produced from SCD HSPCs, and to evaluate the efficacy of the best-performing LV in rescuing the SCD phenotype. The second part of this thesis aimed to develop a novel genome editing-based strategy to restore fetal γ-globin genes expression. This therapeutic approach stems from the observation that the clinical course of β-thalassemia and SCD is improved in the presence of elevated HbF levels. By using the innovative CRISPR/Cas9 technology, we aimed at disrupting repressors binding sites in the γ-globin promoters to reactivate HbF expression in SCD HSPCs-derived RBCs. Reactivating fetal γ-globin genes at their endogenous genomic locus can circumvent the difficulties associated with the relatively low LV-derived transgene expression per vector copy, likely because the low LV vector capacity allows the usage only of short DNA stretches from the LCR, arranged in a non-physiological manner. In addition, this strategy offers a potentially safer targeted approach compared to the LV-based gene addition. In SCD, this therapeutic approach can favor the anti-sickling γ-globin expression, at the expense of the mutated βS-globin, given the competition between the fetal and the adult genes for the interaction with the LCR. In a comparative approach, we intended to evaluate novel and known therapeutic targets in the γ-globin promoters. To this purpose, several gRNAs have been designed to target 3 regions of the γ-globin promoters, where variants associated with elevated HbF levels and/or binding sites for HbF repressors have been described. We aimed to screen these gRNAs in an adult erythroid cell line (HUDEP-2) and SCD HSPCs-derived RBCs in terms of HbF reactivation and correction of the patient phenotype, to select the best therapeutic target for an efficient and safe therapeutic approach for β-hemoglobinopathies

Les malformations du cortex cérébral (MDC) représentent une cause importante de handicap et d'épilepsie pharmaco-résistante. Le séquençage à haut débit a permis une amélioration considérable de l'identification des bases moléculaires des MDC non syndromiques. Toutefois, certaines formes, notamment les MDC complexes, demeurent inexpliquées. Mon projet de thèse a pour objectif de progresser dans la compréhension des MDC complexes en utilisant deux modèles : les microlissencéphalies (MLIS) et le syndrome d'Aicardi (AIC), une forme syndromique particulière associant des malformations de l'oeil et du cerveau uniquement rapporté chez les filles. L'étude par séquençage d'exome en trios de 16 familles MLIS m'a permis d'identifier et de caractériser un nouveau gène, WDR81, impliqué dans le cycle cellulaire. Par la même stratégie, j'ai pu identifier un variant homozygote pathogène dans TLE1, un partenaire majeur de FOXG1 dans la balance prolifération/différenciation de progéniteurs neuronaux, dans une famille consanguine de microcéphalie postnatale dont le phénotype est proche du syndrome FOXG1. En parallèle, mes travaux ont permis de préciser les spectres phénotypiques associés à RTTN, EPG5, COL4A1, COL4A2, TBR1, KIF5C, KIF2A et FOXG1. La deuxième partie de mon projet avait pour objet l'identification des bases moléculaires du syndrome d'Aicardi à partir d'une cohorte internationale de 19 patientes. Après avoir exclu un biais d'inactivation du chromosome X et la présence de microremaniements chromosomiques, j'ai réalisé un séquençage d'exome en trio. Aucun variant récurrent n'a été retrouvé dans les séquences codantes. Dans un second temps, j'ai testé une approche combinant les données du séquençage de génome et l'analyse du transcriptome (RNA-Seq) sur fibroblastes, me permettant d'identifier des transcrits dérégulés qui étaient impliqués dans le développement du cerveau et de l'oeil. J'ai comparé les résultats de cette analyse avec ceux de l'analyse du génome dans le but d'identifier des variants dans ces gènes candidats. En conclusion, mon travail de thèse a permis d'améliorer la connaissance des bases moléculaires des MDC complexes et d'ouvrir des perspectives de nouveaux mécanismes tels que ceux engageant les gènes WDR81 et EPG5, et le rôle des endosomes et de l'autophagie dans les MDC, et aussi TLE1 comme nouvelle cause de microcéphalies postnatales. Mes travaux ont également permis de générer une collection de données de séquençage haut débit (WES, WGS et RNA-Seq) qui seront mises en commun dans le cadre d'un consortium international afin de développer des nouvelles stratégies d'analyse en particulier pour les séquences non codantes. Cette approche permettra également d'ouvrir la voie vers la compréhension des mécanismes cellulaires impliqués dans la formation du cerveau et de l' œil
Malformations of cortical development (MCD) are a major cause of intellectual disability and drug-resistant epilepsy. Next Generation Sequencing (NGS) has considerably improved the identification of the molecular basis of non-syndromic MCD. However, certain forms, including complex MCD, remain unexplained. My PhD project aimed to improve the understanding of complex MCD using two disorders: Microlissencephaly (MLIS) and Aicardi Syndrome (AIC), the latter associating brain and eye malformations and only reported in girls. Trio Whole Exome Sequencing (WES) performed in 16 MLIS families allowed me to identify and functionally characterize a new MLIS gene, WDR81, in which mutations lead to cell cycle alteration. Moreover, using the same strategy, I was able to identify a pathogenic homozygous variant in TLE1 in a patient from consanguineous family with a postnatal microcephaly, suggestive of a FOXG1-like presentation. Interestingly, TLE1 is a major partner of FOXG1, a gene involved in maintaining the balance between progenitor proliferation and differentiation. In parallel, my work allowed me to redefine the phenotypic spectrum associated with RTTN, EPG5, COL4A1 and COL4A2, TBR1, KIF5C, KIF2A and FOXG1. The second part of my PhD program was aimed at identifying the genetic basis of AIC in an international cohort of 19 patients. After excluding a skewed X chromosome inactivation and the presence of chromosomal rearrangements, I performed WES in trios. The analysis of the data from WES did not allow me to identify any recurrent variants. I therefore tested a new approach combining Whole Genome Sequencing (WGS) and RNA-Sequencing (RNA-Seq) on fibroblast cells. I identified a number of deregulated transcripts implicated in brain and eye development. I compared the results of this analysis with the WGS analysis in order to find variants in these candidate genes. In conclusion, these studies have improved the knowledge of the molecular basis of complex MCD, such as TLE1 in postnatal microcephaly, and revealed the pathogenic mechanisms such as WDR81 in cell cycle progression and EPG5 in endosomes and autophagy. My work has also generated a collection of NGS data (WES, WGS and RNA-Seq) that will be shared in an international consortium to develop new analytical strategies, in particular for the non-coding DNA regions. This novel strategy provides opportunities to improve understanding of the cellular mechanisms involved in brain and eye development

Thesis (M.S.)--University of Wisconsin--Madison, 1999.
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Indiana University-Purdue University Indianapolis (IUPUI)
Down syndrome (DS) and Fetal Alcohol Syndrome (FAS) are two leading causes of birth defects with phenotypes ranging from cognitive impairment to craniofacial abnormalities. While DS originates from the trisomy of human chromosome 21 and FAS from prenatal alcohol consumption, many of the defining characteristics for these two disorders are stunningly similar. A survey of the literature revealed over 20 similar craniofacial and structural deficits in both human and mouse models of DS and FAS. We hypothesized that the similar phenotypes observed are caused by disruptions in common molecular or cellular pathways during development. To test our hypothesis, we examined morphometric, genetic, and cellular phenotypes during development of our DS and FAS mouse models at embryonic days 9.5-10.5. Our preliminary evidence indicates that during early development, dysregulation of Dyrk1a and Rcan1, cardinal genes affecting craniofacial and neurological precursors of DS, are also dysregulated in embryonic FAS models. Furthermore, Caspase 3 was also found to have similar expression in DS and FAS craniofacial neural crest derived tissues such as the first branchial arch (BA1) and regions of the brain. This may explain a developmental deficit by means of apoptosis. We have also investigated the expression of pAkt, a protein shown to be affected in FAS models, in cells located within the craniofacial precursor of Ts65Dn. Recent research shows that Ttc3, a gene that is triplicated and shown to be overexpressed in the BA1 and neural tube of Ts65Dn, targets pAkt in the nucleus affecting important transcription factors regulating cell cycle and cell survival. While Akt has been shown to play a role in neuronal development, we hypothesize that it also affects similar cellular properties in craniofacial precursors during development. By comparing common genotypes and phenotypes of DS and FAS we may provide common mechanisms to target for potential treatments of both disorders. One of the least understood phenotypes of DS is their deficient immune system. Many individuals with DS have varying serious illnesses ranging from coeliac disease to respiratory infections that are a direct result of this immunodeficiency. Proteasomes are an integral part of a competent and efficient immune system. It has been observed that mice lacking immunoproteasomes present deficiencies in providing MHC class I peptides, proteins essential in identifying infections. A gene, Psmg1 (Dscr2), triplicated in both humans and in Ts65Dn mice, is known to act as a proteasome assembly chaperone for the 20S proteasome. We hypothesized that a dysregulation in this gene promotes a proteasome assembly aberration, impacting the efficiency of the DS immune system. To test this hypothesis we performed western blot analysis on specific precursor and processed β-subunits of the 20S proteasome in thymic tissue of adult Ts65Dn. While the β-subunits tested displayed no significant differences between trisomic and euploid mice we have provided further insight to the origins of immunodeficiency in DS.

The lung is an organ that contains a vast system of airways carefully constructed to achieve maximal surface area in a confined space, requiring guidance from a multitude of developmental factors. The Shh pathway is one such signaling mechanism that is critical to proper lung formation, guiding branching morphogenesis and cellular proliferation through its downstream Gli transcription factors. Additionally, Foxf1 has been shown to be a key developmental factor required for proper lung formation during embryogenesis. Although theorized that the Gli transcription factors are responsible for regulating foxf1 levels, their exact relationship has yet to be revealed. Using five different models for Shh signaling (gli2 null, gli2 over-expressor [hVER-Gli2], gli3 null, Gli3 constitutive repressor [Gli3Δ699] and cyclopamine treated lung explants), I compared and contrasted the role of Gli2 and Gli3 in terms of their effect on cell cycle regulation, and on the expression levels of foxf1 and its potential downstream target genes tbx4, tbx5 and fgf10. I found that ectopic over-expression of gli2 resulted in increased Shh pathway activation, and increased expression of G1/S phase cyclins, which was associated with increased cellular proliferation and lung growth. However, no change in the levels of G1/S phase cyclins due to altered Gli3 signaling was observed. Foxf1 levels positively correlate with the levels of gli2, and appear to be independent of Gli3 activity. The amount of tbx4, tbx5, and fgf10 transcripts were observed to follow the levels of gli2 in the different gli2 mouse models, however, there was no significant change in gli3 null or Gli3Δ699 mice. Finally, by analyzing gene expression at different time points during gestation, I found that while gli2 levels affect foxf1 throughout gestation, the relationship to tbx4, tbx5 and fgf10, occurs only during the latter stages of lung development. I conclude, that Gli2 and not Gli3 appears to be the primary transducer of Shh signaling influencing cyclin regulation, leading to changes in embryonic lung growth. Furthermore, that Gli2 and not Gli3 appears to regulate foxf1 expression levels, and that this may extend downstream to influence tbx4, tbx5 and fgf10 expression.

Intrauterine and perinatal factors have been linked to risk of testicular cancer and breast cancer in the offspring. In particular, many studies have provided evidence that supports the hypothesis of an intrauterine component in the origin of breast cancer. Human studies to examine the underlying biological mechanisms, however, have been limited. In Chapter 1, we review the likely role of stem cells in hormone-mediated carcinogenic process, particularly as intermediate steps between in utero exposure to hormones and breast cancer. We summarize also studies related to the assumptions of the hypothesis concerning in utero exposure. In Chapter 2 and 3, we report a reproducibility study for hormone assay and a population-based study that measured stem cell potential to explore mechanisms mediating the relation between in utero exposure to pregnancy hormones and cancer risk in the offspring. In these studies, we utilized umbilical cord blood collected two collection sites. Cord blood donors were 99 women, 18 years or older, who delivered a singleton birth. We assayed plasma concentrations of estradiol, unconjugated estriol, testosterone, progesterone, prolactin, sex-hormone binding globulin, insulin-like growth factor-1 (IGF-1), and IGF binding protein-3 (IGFBP-3). We observed in a reproducibility study that assays of these plasma hormones and binding proteins are reliable. For stem cell potential, we measured concentrations of CD34+, CD34 +CD38− cells, CD34+c-kit +. We applied linear regression analysis and controlled for maternal and neonatal characteristics. Data from two sample collection sites were analyzed separately and collectively. Based on the pooled data further taking into account cord sample collection sites, we found that CD34+ increases with increasing E3 (23% per SD increase, p = 0.02) and IGF-1 (25% per SD increase, p = 0.04). A similar increase was observed for IGFBP-3 (p = 0.05). E3, T, SHBG, and progesterone were weakly associated with CD34+CD38 −. These findings indicate that levels of growth factors and steroid hormones are associated with stem cell potential in human umbilical cord blood and point to a potential mechanism that may mediate the relation between in utero exposure to hormones and cancer risk in the offspring.

Zhang, Siwei.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 87-98).
Abstracts in English and Chinese.
Chapter 1. --- General introduction --- p.1
Chapter 1.1. --- "Types, structure and function of human hemoglobin" --- p.1
Chapter 1.1.1. --- Structure and functions of human hemoglobin --- p.1
Chapter 1.1.2. --- Types of human hemoglobin --- p.2
Chapter 1.2. --- Regulatory mechanism of human hemoglobin expression --- p.3
Chapter 1.2.1. --- The human a and β locus --- p.3
Chapter 1.2.2. --- Development of globin genes switching concept --- p.4
Chapter 1.2.3. --- Factors that regulate globin gene expression --- p.5
Chapter 1.2.3.1. --- The locus control region (LCR) --- p.5
Chapter 1.2.3.2. --- The cis-regulatory elements --- p.5
Chapter 1.2.3.3. --- The trans-acting factors --- p.6
Chapter 1.3. --- The human hemoglobinopathies --- p.8
Chapter 1.3.1. --- α-thalassemia --- p.8
Chapter 1.3.2. --- β-thalassemia --- p.9
Chapter 1.3.3. --- Sickle cell anemia --- p.10
Chapter 1.4. --- Current approaches towards β-thalassemia treatment --- p.11
Chapter 1.4.1. --- Blood transfusion --- p.11
Chapter 1.4.2. --- Bone marrow transplantation --- p.12
Chapter 1.4.3. --- Drug-induced activation of fetal hemoglobin production --- p.12
Chapter 1.4.3.1. --- Hydroxyurea --- p.12
Chapter 1.4.3.2. --- Butyrate and short-chain fatty acids --- p.13
Chapter 1.4.3.3. --- "Mutagens, DNA methyltransferase inhibitors and other HbF inducible agents" --- p.13
Chapter 1.4.3.4. --- Cucurbitacin D --- p.14
Chapter 1.4.4. --- Gene therapy --- p.14
Chapter 1.5. --- Research Objectives --- p.15
Chapter 2. --- "Analysis of CuD, Hydroxyurea and other inducers on the induction of α, β, γ, δ， ε，ζ BP-1 genes and fetal hemoglobin induction" --- p.16
Chapter 2.1. --- Introduction --- p.16
Chapter 2.1.1. --- Properties of human K562 cell line --- p.16
Chapter 2.1.2. --- Induction and measurement of fetal hemoglobin --- p.16
Chapter 2.1.3. --- "Induction of α, β, γ, δ, ε , ζ and BP-1 gene and Real-time RT-PCR analysis" --- p.17
Chapter 2.2. --- Materials --- p.18
Chapter 2.2.1. --- Chemicals and reagents --- p.18
Chapter 2.2.2. --- Kits --- p.19
Chapter 2.2.3. --- Buffers and solutions --- p.19
Chapter 2.2.4. --- Cell lines --- p.20
Chapter 2.3. --- Experimental procedures --- p.20
Chapter 2.3.1. --- Hemoglobin quantity measurement by HbF ELISA --- p.20
Chapter 2.3.1.1. --- MTT assay --- p.21
Chapter 2.3.1.2. --- Preparation of capture-antibody coated ELISA plates --- p.21
Chapter 2.3.1.3. --- Plate blocking --- p.22
Chapter 2.3.1.4. --- Sample and standard preparation --- p.22
Chapter 2.3.1.5. --- HRP antibody and colorimetric detection --- p.23
Chapter 2.3.1.6. --- Statistical analysis --- p.23
Chapter 2.3.2. --- Preparation of mRNA extract from K562 cells --- p.23
Chapter 2.3.3. --- Reverse transcription and Real-time PCR analysis --- p.24
Chapter 2.4. --- Results --- p.25
Chapter 2.4.1. --- CuD significantly upregulates HbF expression in K562 cells --- p.25
Chapter 2.4.2. --- "CuD augments α, β, γ, δ, ε , ζ and BP-1 genes at different level in K562 cells" --- p.28
Chapter 2.4.3. --- Cucurbitacin D-induced γ-globin gene activation requires12-24 hours in K562 cells --- p.31
Chapter 2.5. --- Discussion --- p.33
Chapter 2.5.1. --- Enhancement of fetal hemoglobin production using different chemical compounds --- p.33
Chapter 2.5.2. --- CuD increased HbF synthesis by increasing γ-globin mRNA amount --- p.35
Chapter 2.5.3. --- CuD and HU down-regulated the BP-1 gene expression --- p.36
Chapter 3. --- Determination of potential signal transduction pathways during CuD and HU-mediated fetal hemoglobin production --- p.36
Chapter 3.1. --- Introductions --- p.36
Chapter 3.1.1. --- The p38 MAPK family --- p.37
Chapter 3.1.2. --- The JAK2-STAT3 pathway --- p.38
Chapter 3.1.3. --- Fundamentals on inhibition assay of p38 MAPK and JAK2-STAT3 pathway --- p.39
Chapter 3.1.4. --- Fundamentals on nuclear translocation of STAT3 --- p.41
Chapter 3.2. --- Materials --- p.41
Chapter 3.2.1. --- Chemicals and reagents --- p.41
Chapter 3.2.2. --- Kits --- p.44
Chapter 3.2.3. --- Buffers and solutions --- p.44
Chapter 3.3. --- Experimental procedures --- p.45
Chapter 3.3.1. --- Detection of p3 8 MAPK phosphorylation status --- p.46
Chapter 3.3.1.1. --- Preparation of cytosolic protein extracts --- p.46
Chapter 3.3.1.2. --- Quantitative measurement of phospho-p38 and pan-p38 by ELIS A method --- p.46
Chapter 3.3.1.2.1. --- Antigen adsorption and establishment of standard curves --- p.46
Chapter 3.3.1.2.2. --- Plate washing and application of detection antibody --- p.47
Chapter 3.3.1.2.3. --- Plate washing and application of secondary antibody --- p.47
Chapter 3.3.1.2.4. --- Plate washing and chromogen detection --- p.48
Chapter 3.3.2. --- Detection of signal cascade on JAK2-STAT3 pathway --- p.48
Chapter 3.3.2.1. --- Preparation of cytosolic protein extracts for Western Blot detection --- p.48
Chapter 3.3.2.2. --- Gel running and Western Blot detection --- p.48
Chapter 3.3.3. --- Quantitative measurement of phospho-STAT3-Tyr705 using ELISA method --- p.50
Chapter 3.3.3.1. --- Preparation of cytosolic protein extracts --- p.50
Chapter 3.3.3.2. --- Reconstitution and Dilution of STAT3 [pY705] Standard --- p.50
Chapter 3.3.3.3. --- Measurement of STAT3 [pY705] concentration in cell lysates --- p.51
Chapter 3.3.4. --- Inhibitor assay of JAK2-STAT3 and p38 MAPK pathway --- p.52
Chapter 3.3.4.1. --- Establishment of inhibitor assay --- p.52
Chapter 3.3.4.2. --- HbF ELISA detection --- p.53
Chapter 3.3.5. --- Detection of STAT3 nuclear translocation and DNA binding affinity --- p.53
Chapter 3.3.5.1. --- Preparation of nuclear extract from K562 cells --- p.53
Chapter 3.3.5.2. --- EMS A detection of transcriptional factors binding to γ-promoter region --- p.54
Chapter 3.3.5.2.1. --- 3´ة end-labeling of EMS A probes --- p.54
Chapter 3.3.5.2.2. --- Dot blotting for labeling efficiency estimation --- p.56
Chapter 3.3.5.2.3. --- EMSA binding reaction and non-denaturing gel electrophoresis --- p.57
Chapter 3.3.5.2.4. --- Membrane development and chemiluminescence detection --- p.58
Chapter 3.3.5.3. --- Preparation of K562 samples for immunofluorescence detection --- p.60
Chapter 3.3.5.3.1. --- Slide coating for cell capture --- p.60
Chapter 3.3.5.3.2. --- Preparation of cell slide --- p.60
Chapter 3.3.5.3.3. --- Sample fixation and antibody probing treatment --- p.60
Chapter 3.3.5.3.4. --- Sample imaging and immunofluorescence detection --- p.61
Chapter 3.4 --- Results --- p.62
Chapter 3.4.1. --- Activation of p38 MAPK pathway and STAT3 phosphorylation by hydroxyurea --- p.62
Chapter 3.4.1.1. --- "The p38 MAPK pathway is activated by hydroxyurea, but not activated by Cucurbitacin D" --- p.62
Chapter 3.4.1.2. --- Increased p38 phosphorylation level elicits STAT3 phosphorylation at Ser727 site --- p.64
Chapter 3.4.2. --- Activation of JAK2 and STAT3 phosphorylation by Cucurbitacin D --- p.66
Chapter 3.4.2.1. --- Cucurbitacin D promotes JAK2 activation --- p.66
Chapter 3.4.2.2. --- Cucurbitacin D and hydroxyurea promote STAT3 phosphorylation at Tyr705 site --- p.66
Chapter 3.4.3. --- Basal activity of signal transduction pathways is essential for HbF induction --- p.69
Chapter 3.4.3.1. --- Activation of γ-globin gene requires presence of basal phosphorylation level of p38 MAPK --- p.69
Chapter 3.4.3.2. --- Inhibition on JAK2-STAT3 pathway results in reduced fetal hemoglobin production --- p.71
Chapter 3.4.4. --- Translocation and DNA binding of STAT under Cucurbitacin D induction --- p.72
Chapter 3.4.4.1. --- Cucurbitacin D and hydroxyurea both enhance binding affinity of transcriptional factors to the Gγ/Aγ promoter --- p.72
Chapter 3.4.4.2. --- Cucurbitacin D and hydroxyurea induces nuclear translocation of STAT3 --- p.75
Chapter 3.5. --- Discussion --- p.77
Chapter 3.5.1. --- The role of p38 MAPK activation during γ-globin gene activation --- p.77
Chapter 3.5.2. --- STAT3 phosphorylation at Ser727 site promotes transcription factor activity and γ-globin gene expression --- p.77
Chapter 3.5.3. --- The role of JAK2-STAT3 activation during γ-globin gene activation --- p.78
Chapter 3.5.4. --- Inhibitor assay --- p.79
Chapter 3.5.5. --- Relations between STAT3 nuclear translocation and enhanced fetal hemoglobin production --- p.82
Chapter 4. --- Summery and Prospect --- p.83
Chapter 5. --- References --- p.87

Maternal consumption of ethanol during pregnancy contributes to a set of pathologies, grouped together as the fetal alcohol spectrum disorders, affecting as many as 5% of live births in the United States annually. Ethanol acts widely in the developing embryo, affecting many tissues, but causing deficits in neuronal and neural crest populations particularly. These deleterious effects cause archetypical craniofacial expression and neurological deficits, including microcephaly and neuronal dysfunction. Severity of symptoms is linked to frequency of maternal alcohol consumption as well as the maximum blood alcohol concentration reached by the mother. The teratology of ethanol has been widely researched over the last four decades, with the link between the neural crest pathology and the fetal alcohol spectrum phenotype becoming clearer. Animal model studies have managed to replicate many of the symptoms seen in humans afflicted with fetal alcohol spectrum disorders, and have allowed us to elucidate the biochemical mechanisms behind the disease. There is no singular pathway responsible for the fetal alcohol spectrum disorders: over half a dozen models of dysfunction have been identified, and ethanol’s ability to react with a series of targets means that more pathways are likely to be discovered. Current theories regarding the effects of ethanol on the neural crest have implicated apoptosis of the cephalic neural crest, mediated by G-protein coupled receptors, activation of a phospholipase C pathway, and subsequent release of intracellular calcium; perturbations of the actin cytoskeleton leading to migration dysfunction of neural crest cells in the developing neural tube; lack of functional trophic molecules, specifically Shh, likely due to dysfunction of the cholesterol biosynthetic pathway; lack of retinoic acid production; oxidative stress, production of reactive oxygen species, and iron dysregulation; and genetics, which seems to confer greater susceptibility and resistance to ethanol in certain individuals. Ultimately, a global model for ethanol’s actions on the developing fetus eludes researchers, as do any potential treatments, and more research is required to further elucidate ethanol’s teratogenic mechanism.

碩士
中興大學
食品暨應用生物科技學系
95
Studies have suggested that higher intakes of lycopene are associated with a reduced risk of several types of cancer, such as prostate cancer, hepatoma and coronary heart disease. Cell culture studies are useful for elucidating the mechanisms of action of lycopene, and tetrahydrofuran (THF) has commonly been used to deliver lycopene to cells. However, the use of THF as the solvent for lycopene has repeatedly been questioned because of its disadvantages in cell culture studies such as THF oxidizes readily in culture media and low cellular association. Therefore, attempts have been made by using vehicles other than THF for delivering lycopene to cells but it also suffers limitations such as cytotoxicity, poor solubility and crystallization in the medium. Here, we first compared the FBS dilution method of lycopene with other solubilization vehicles for lycopene including THF, THF containing 0.0025% butylated hydroxytoluene (THF/BHT), liposome, methyl-beta-cyclodextrin (M-β-CD) and micelles in cultured cells. We investigated that lycopene in FBS led to significantly higher stability and cellular uptake of lycopene than that in THF, THF/BHT, liposome or M-β-CD. Furthermore, when FBS was replaced with lipoprotein-deficient serum, the uptake of lycopene by DU145 cells was markedly decreased and was not significantly different from that of THF or THF/BHT. The results strongly indicate that the lipoprotein fraction of FBS plays an important role in delivering lycopene to cells. We then went on to study the improvement the methods of use THF/FBS as delivery vehicle because the stability of lycopene in this method is not satisfactory. Furthermore, the cellular uptake of lycopene was positively correlated with the degradation rate of lycopene in different delivery vehicles (r2 = 0.71) during incubation times (data not shown). We therefore used various antioxidants that may protect lycopene to decrease the degradation rate of lycopene in cultured studies. We found that use THF/FBS as delivery vehicle combined with quercetin (10µM) can better stabilize lycopene than can other antioxidants combining in this study, and similar result also suggested in cellular uptake efficiency. We further adopted the method of THF/FBS combining with quercetin in dose-dependent manners. When combining with 50 µM quercetin, the lycopene remained and cellular uptake was higher than that with 10 and 30 µM. In summary, this thesis demonstrated that the use of FBS for delivering lycopene into the two prostate cancer cell lines is superior to the use of THF, THF/BHT, liposome, M-β-CD and micelle and that the lipoprotein of FBS is likely responsible for the improved stability and cellular uptake of lycopene. Furthermore, antioxidants especially quercetin improves the use of THF/FBS as vehicle for delivering lycopene by increasing cellular uptake and increasing stability of lycopene by preventing lycopene oxidation. We conclude that using THF/FBS as vehicle for delivering lycopene combined with quercetin may contribute relatively to the in vitro studies of lycopene in the future.

幹細胞療法是近年的研究熱點之一，然而幹細胞在組織修復中的實際應用受到移植後幹細胞存活率低的制約，約80% 的幹細胞在移植至組織後不能存活。 人類臍帶血管周皮 (HUCPV) 幹細胞為多功能間充質幹細胞移植提供豐富的細胞來源。 在合適的誘導環境下，它們具有向多種間充質細胞系分化的能力。 與從骨髓或臍帶血中提取的間充質幹細胞比較，人類臍帶血管周皮幹細胞的體外增殖更為容易。 在本研究中，我們從人類臍帶血管周圍組織中分離人類臍帶血管周皮幹細胞，並採用流式細胞技術分選細胞表面標記物CD34、CD45呈陰性同時CD44 、CD90、 CD105、 CD146呈陽性的HUCPV細胞。HUCPV細胞在體外培養以及三維支架的環境下具有分化為骨和軟骨的能力。
在本研究中，我們主要研究腦和生殖器官表達基因（BRE）在HUCPV細胞中的功能。 BRE蛋白與其他已知蛋白的同源性均不高，目前尚未鑑定出任何功能性的結構域。 至今為止，BRE基因的已知功能大多數是通過對腫瘤模型的研究發現的。 據報導，BRE能夠提高DNA損傷的腫瘤細胞的存活率，但BRE在幹細胞中的作用仍不清楚。 我們發現，當HUCPV細胞分化後，其BRE的表達水平降低。 此外，利用BRE-siRNA降低HUCPV細胞中BRE基因的表達，能夠促進HUCPV細胞向骨和軟骨分化的進程。 因此，我們假設BRE對維持HUCPV細胞的幹細胞功能具有重要的作用。 由於經過BRE基因沉默處理的HUCPV細胞與對照組相比並無顯著的表型差別，我們採用微陣列（microarray）以及比較蛋白組學的方法研究兩者間的區別，從而找出BRE基因的功能以及可能涉及BRE的信號通路。
通過微陣列技術，我們深入地分析了BRE基因表達沉默後HUCPV細胞的轉錄組。 在經過BRE基因沉默處理的HUCPV細胞中，我們發現與維持幹細胞多向分化潛能有關的OCT4、 FGF5和FOXO1A等基因的表達顯著下調。 另外，BRE基因的沉默能夠影響表觀遺傳調控基因以及TGF-β 信號通路組成部件的表達，而TGF -β 信號通路是維持幹細胞自我更新的重要通路。 這些結果提示，BRE作為一個重要的調控因子，在維持HUCPV細胞的多向分化潛能的同時能夠防止細胞分化。
在比較蛋白組學的研究中，我們發現BRE基因的沉默能夠降低細胞骨架結合蛋白的表達，例如actin, annexin II 及 tropomyosin。 此外，我們利用免疫共沉澱的方法證明了BRE蛋白與actin及 annexin II蛋白直接結合。 細胞骨架的改變可能為HUCPV細胞的分化提供了一個有利的環境，因而BRE基因的沉默能夠促進HUCPV細胞向骨和軟骨分化。 支持這一推論的其中一個依據是Lim et al., 2000; Solursh, 1989; Zhang et al., 2006，文獻報導肌動蛋白多聚化抑製劑能夠促進軟骨形成的過程。 綜上所述，本研究為進一步研究BRE基因在HUCPV細胞中的功能以及與BRE直接作用的蛋白打下了基礎。
Stem cells therapy has gained considerable attention in recent years. However, the practical use of stem cells for tissue repair has been hindered due to their low survival rate after grafting into tissues, for approximately 80% of the stem cells died after implantation. Human umbilical cord perivascular (HUCPV) stem cells offer a new and rich resource of multipotent mesenchymal stem cells. These cells possess the ability to differentiate into various mesenchymal cell lineages when induced. HUCPV cells can be more easily amplified in culture than mesenchymal stem cells extracted from bone marrow or umbilical cord blood. In this study, HUCPV cells were isolated from the perivascular regions of human umbilical cords. The HUCPV cells were sorted using flow cytometer for CD34⁻, CD44⁺, CD45⁻, CD90⁺, CD105⁺ and CD146⁺ surface markers. These HUCPV cells were found to be capable of differentiating into osteogenic lineage in monolayer culture and chondrogenic lineage in pellet culture. These cells were also found to be capable of differentiating into osteogenic and chondrogenic lineage in silk fibroin which acted as three-dimensional scaffolds for the cells to grow on.
The function of the Brain and Reproductive Organ-Expressed (BRE) gene in the context of HUCPV cells was investigated. The BRE protein shares no homology with any other known gene products and contains no known functional domain. To date, most of what we know about the function of this gene has been conducted in the tumor model. It has been reported that BRE can enhance the cellular survival of cancer cells following DNA damage. The role of BRE in stem cells has never been examined. We have established that BRE expression was down-regulated when HUCPV cells started to differentiate. In addition, silencing BRE expression, using BRE-siRNA, in HUCPV cells could accelerate osteogenic and chondrogenic differentiation. Hence, we hypothesized that BRE played an important role in maintaining the stemness of HUCPV cells. Because there was a lack of phenotypic difference between the BRE-silenced HUCPV cells and cells transfected with the control-siRNA, we decided to profile these cells using microarray and proteomic analyses. The aim was to elucidate the function of the BRE gene and establish whether BRE was involved in any signaling pathways.
In the microarray analysis, we examined the transcriptome of HUCPV cells in response to BRE-silencing in depth. Amongst the genes that we identified were significantly down-regulated by BRE-silencing and involved in the maintenance of pluripotency in ES cells were OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and also components of the TGF-β signaling pathway. This pathway is crucially involved in maintaining stem cell self-renewal. Therefore, we propose that BRE acts like a modulator that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.
In the comparative proteomic study, BRE-silencing resulted in decreased expression patterns of cytoskeletal binding proteins such as actin, annexin II and tropomyosin. In addition, co-immunoprecipitation experiments revealed that the BRE protein can bind directly with actin and annexin II. It is possible that altering the cytoskeleton may provide a favorable environment for HUCPV cells to differentiate. This may explain why we were able to accelerate osteogenic and chondrogenic differentiation following BRE-silencing. In support of the view, it has been reported that chondrogenesis could be enhanced after cells have been treated with actin polymerization inhibitors (Lim et al., 2000; Solursh, 1989; Zhang et al., 2006). In sum, our studies provide an insight into the function of the BRE gene in HUCPV cells and the proteins that BRE can directly act on.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Chen, Elve.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 135-159).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Thesis/Assessment Committee --- p.i
Abstract --- p.ii
摘要 --- p.v
Acknowledgements --- p.viii
List of Figures --- p.ix
List of Tables --- p.xiii
Table of Abbreviations --- p.xiv
Contents --- p.xviii
Chapter 1 --- p.1
Literature Review --- p.1
Chapter 1.1 --- Stem cells --- p.1
Chapter 1.2 --- Embryonic stem cells (ESCs) --- p.2
Chapter 1.3 --- Epiblast-derived stem (EpiS) cells --- p.2
Chapter 1.4 --- Somatic stem cells (SSCs) --- p.3
Chapter 1.5 --- Induced pluripotent stem (iPS) cells --- p.5
Chapter 1.6 --- Human umbilical cord perivascular (HUCPV) cells --- p.7
Chapter 1.7 --- CD146 --- p.8
Chapter 1.8 --- Stem cell senescence --- p.9
Chapter 1.9 --- Brain and reproductive organ-expressed (BRE) protein --- p.12
Chapter 1.10 --- Stem cell self-renewal --- p.14
Chapter 1.11 --- Apoptosis --- p.16
Chapter 1.12 --- Stem cell niche --- p.21
Chapter 1.13 --- Stem cell homing --- p.22
Chapter 1.14 --- Objective --- p.22
Chapter 2 --- p.24
Accelerated osteogenic and chondrogenic differentiation of HUCPV cells by modulating the expression of BRE --- p.24
Chapter 2.1 --- Introduction --- p.24
Chapter 2.2 --- Rationale --- p.27
Chapter 2.3 --- Materials and Methods --- p.27
Chapter 2.3.1 --- Extraction of HUCPV cells from umbilical cord --- p.27
Chapter 2.3.2 --- Cell culture condition --- p.28
Chapter 2.3.3 --- Flow cytometry analysis and cell sorting --- p.28
Chapter 2.3.4 --- In vitro osteogenic differentiation --- p.29
Chapter 2.3.5 --- In vitro chondrogenic differentiation --- p.29
Chapter 2.3.6 --- Alcian blue staining --- p.29
Chapter 2.3.7 --- Alizarin red S staining --- p.30
Chapter 2.3.8 --- Immunofluorescence analysis --- p.30
Chapter 2.3.9 --- Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) --- p.31
Chapter 2.3.10 --- Transfection with siRNA --- p.35
Chapter 2.3.11 --- Microarray --- p.35
Chapter 2.3.12 --- Cell lysis and immunoprecipitation --- p.36
Chapter 2.3.13 --- SDS-PAGE and Western blot --- p.36
Chapter 2.3.14 --- Isoelectric focusing and 2-dimensional gel electrophoresis --- p.37
Chapter 2.3.15 --- Migration (wound healing) assay --- p.38
Chapter 2.4 --- Results --- p.38
Chapter 2.4.1 --- HUCPV cells were capable to differentiate into osteoblasts and chondrocytes --- p.38
Chapter 2.4.2 --- BRE expression is down-regulated when HUCPV cells begins to differentiate --- p.40
Chapter 2.4.3 --- Silencing of BRE expression accelerates induction of osteogenesis and chondrogenesis --- p.40
Chapter 2.4.4 --- Microarray analysis of BRE-silenced HUCPV cells --- p.42
Chapter 2.4.4.1 --- Stemness factors --- p.43
Chapter 2.4.4.2 --- Epigenetic regulation --- p.43
Chapter 2.4.4.3 --- Signaling pathways crucial for stemness maintenance --- p.44
Chapter 2.4.4.4 --- TGF-β signaling --- p.44
Chapter 2.4.4.5 --- FGF signaling --- p.44
Chapter 2.4.4.6 --- NOTCH signaling --- p.45
Chapter 2.4.4.7 --- WNT signaling --- p.46
Chapter 2.4.4.8 --- Homeobox transcription factors (HOX) --- p.46
Chapter 2.4.4.9 --- Cell cycle regulation --- p.47
Chapter 2.4.4.10 --- Chemokines and cytokines regulation --- p.48
Chapter 2.4.4.11 --- Apoptosis --- p.49
Chapter 2.4.5 --- BRE-silencing alters the cellular proteome of HUCPV cells --- p.50
Chapter 2.4.5.1 --- BRE-silencing alters the cytoskeletal binding proteins of HUCPV cells --- p.51
Chapter 2.4.5.2 --- BRE-silencing alters the expressions of stemness-related proteins in HUCPV cells --- p.52
Chapter 2.4.5.3 --- BRE-silencing alters the expressions of apoptosis-related proteins in HUCPV cells --- p.53
Chapter 2.5 --- Discussion --- p.86
Chapter 2.5.1 --- Microarray study discussion --- p.87
Chapter 2.5.2 --- Proteomic study discussion --- p.89
Chapter 3 --- p.93
Replicative senescence alters the transcriptome and proteome of HUCPV cells --- p.93
Chapter 3.1 --- Introduction --- p.93
Chapter 3.2 --- Materials and methods --- p.93
Chapter 3.3 --- Results --- p.93
Chapter 3.3.1 --- Microarray analysis of aged HUCPV cells --- p.94
Chapter 3.3.1.1 --- Stemness factors --- p.95
Chapter 3.3.1.2 --- Epigenetic regulation --- p.96
Chapter 3.3.1.3 --- Senescence associated markers --- p.96
Chapter 3.3.1.4 --- Chemokines and cytokines regulation --- p.97
Chapter 3.3.1.5 --- Matrix metalloproteinases regulation --- p.97
Chapter 3.3.1.6 --- WNT signaling --- p.98
Chapter 3.3.1.7 --- Toll-like receptor signaling pathway --- p.98
Chapter 3.3.2 --- Proteomic profiling of aged HUCPV cells --- p.98
Chapter 3.4 --- Discussion --- p.117
Chapter 3.4.1 --- Aging alters the transcriptome of HUCPV cells --- p.117
Chapter 3.4.2 --- Aging alters the proteome of HUCPV cells --- p.118
Chapter 4 --- p.121
Osteogenic and chondrogenic differentiation capacities of HUCPV cells in silk fibroin scaffold --- p.121
Chapter 4.1 --- Introduction --- p.121
Chapter 4.2 --- Materials and methods --- p.121
Chapter 4.2.1 --- Extraction of silk fibroin --- p.121
Chapter 4.2.2 --- Fabrication of porous silk fibroin scaffold --- p.122
Chapter 4.2.3 --- Scanning electron microscopy --- p.123
Chapter 4.2.4 --- Cell culture --- p.123
Chapter 4.3 --- Results --- p.124
Chapter 4.4 --- Discussion --- p.132
Chapter 5 --- p.133
Conclusions --- p.133
References --- p.135

Indiana University-Purdue University Indianapolis (IUPUI)
Human cord blood (CB) derived circulating endothelial colony forming cells (ECFCs) display a hierarchy of clonogenic proliferative potential and possess de novo vessel forming ability upon implantation in immunodeficient mice. Since survival of ECFC post-implantation is a critical variable that limits in vivo vasculogenesis, we tested the hypothesis that activation of Notch signaling or co-implantation of ECFC with human platelet lysate (HPL) would enhance cultured ECFC vasculogenic abilities in vitro and in vivo. Co-implantation of ECFCs with Notch ligand Delta-like 1 (DL1) expressing OP9 stromal cells (OP9-DL1) decreased apoptosis of ECFC in vitro and increased vasculogenesis of ECFC in vivo. The co-culture of ECFC with HPL diminished apoptosis of ECFC by altering the expression of pro-survival molecules (pAkt, pBad and Bcl-xL) in vitro and increased vasculogenesis of human EC-derived vessels both in vitro and in vivo. Thus, activation of the Notch pathway by OP9-DL1 stromal cells or co-implantation of ECFC with HPL enhances vasculogenesis and augments blood vessel formation by diminishing apoptosis of the implanted ECFC. The results from this study will provide critical information for the development of a cell therapy for limb and organ re-vascularization that can be applied to recovery of ischemic tissues in human subjects.

Aujourd'hui, l'un des modèles de souris humanisées le plus robuste est obtenu en injectant des cellules souches hématopoïétiques humaines (HSC) issues de foie fœtal humain et en implantant du thymus fœtal autologue. Ce modèle, appelé BLT (Bone marrow/Liver/Thymus), s'est révélé capable de supporter une reconstitution, une maturation et une sélection optimales des cellules T. Les souris BLT sont utilisées pour de nombreuses études telles que la compréhension de la biologie du VIH ou plus récemment en médecine régénérative. Grâce à ce modèle, nous avons pu d’une part étudier le rôle des cellules dendritiques plasmacytoïdes (pDC) lors de l’infection par le VIH mais aussi mieux comprendre la formation in vivo de tératomes lors de l’utilisation d’iPSC. Cependant, l'une des principales limites de cette technique réside dans l'obtention du tissu fœtal. Ici, nous avons décrit un nouveau protocole de souris humanisées greffées avec du thymus humain en utilisant des matériaux plus accessibles: du thymus humain retiré lors d’une chirurgie cardiaque chez des nouveaux-nés ou des enfants, et des HSC de sang de cordon. Des morceaux de ces thymus ont été implantés dans les quadriceps de souris immunodéficientes, après avoir été mis en culture. Ces souris CCST (Cord blood and Cardiac Surgery Thymus) ont permis une prise de greffe importante et un meilleur développement des lymphocytes T humains que les souris humanisées sans thymus. Les lymphocytes T des souris CCST et BLT ont montré une fonction similaire, évaluée par des tests de prolifération ex vivo et par rejet de lignées de cellules leucémiques allogéniques in vivo. Nous avons testé l’intérêt de cette nouvelle stratégie dans le modèle de l’infection au VIH-1, qui représente le modèle type de l’utilité des BLT. Nous avons montré que les souris CCST sont sensibles à l'infection par le VIH-1 par voie muqueuse ou intrapéritonéale, comme l'indique la détection de l'ADN du VIH et des cellules p24 +, similairement aux souris BLT. Les souris CCST ont présenté des réponses de lymphocytes T spécifiques du VIH-1 ex vivo plus efficaces que les BLT. Lors du traitement antirétroviral, les souris CCST, comme les BLT, ont vu leur charge virale diminuer. Ces résultats démontrent que les souris CCST représentent une alternative au modèle de souris BLT classique. Ces thymus, éthiquement plus facile à obtenir, peuvent être utilisés pour générer un grand nombre de souris par rapport aux thymus fœtaux.
Immunodeficient mice engrafted with human immune system provide an exciting in vivo model for a better understanding of its functioning and for development of new therapies. Today, one of the most robust humanized mouse model is achieved by injecting human hematopoietic stem cells (HSC) from fetal liver along with an implantation of autologous fetal thymic tissue. This model, called BLT, was shown to be able to support an optimal T cell reconstitution, maturation and selection. BLT mice are extensively used for many studies such as understanding HIV biology or in regenerative medicine. Indeed, our work used BLT mice on one hand to study the role of plasmacytoid dendritic cells (pDC) during the HIV infection and on the other hand to better understand the formation of teratomes from iPSCs in vivo. However, one of the biggest limitations of this technique is the procurement of the fetal tissue. Here we describe a new protocol to do humanized mice engrafted with human thymus pieces by using more accessible materials: human thymus obtained during cardiac surgery and cord blood HSC. Indeed, thymus is spontaneously removed during cardiac surgery in neonates and young children, thus it is an easy and ethical way to obtain this tissue. Those thymuses pieces were implanted in the quadriceps of a immunodeficient mice, after being put in culture. CCST mice (Cord blood and Cardiac Surgery Thymus) exhibited a significant engraftment of T-cells, compared to humanized mice without thymus. T-cells from both CCST and BLT mice showed a similar function as evaluated by proliferation assays upon PHA stimulation ex vivo and rejection of allogeneic leukemic cells lines in vivo. CCST mice were susceptible to HIV-1 infection via mucosal or intraperitoneal route, as shown by detectable viral load, HIV DNA and p24+ cells, at similar levels to those of BLT mice. Importantly, CCST mice displayed more effective ex vivo HIV-1-specific T-cell responses compared to BLT. Upon antiretroviral treatment, CCST mice, like BLT, were able to diminish the viral load. Our data suggest that CCST mice represent an alternative to the regular BLT mouse model. Those easy-to-access thymuses can be used to generate a large number of mice compared to fetal thymuses.