Дисертації з теми "Fibroblasts reprogramming"

The Piwil2 gene belongs to the Piwi family of genes conserved during evolution from Arabidopsis to human. The Piwi family genes are considered as stem cell modulators functioning in meristem cell division in Arabidopsis and germ stem cell propagation in C.elegans and in mammals. Although some essential functions such as germ cell development, transposition repression, epigenetic modification and translational regulation, as well as stem cell and cancer stem cell maintenance, are attributed to this family of genes, the detailed mechanism of the function of these genes still remains elusive. In this study, by taking advantage of the gam of function technique, the coding sequence of the Piwil2 gene was introduced into fibroblasts and stable Piwil2 expressing fibroblasts were established. These cells were evaluated for expression of germ cell specific markers, since the Piwil2 gene is well known as a regulator of spermatogenesis. Piwil2 transfected cells did not express specific markers of spermatogenesis, except Stra8 and Fragilis, but they did express the pluripotent markers of Oct4, Nanog, c-Myc and Klf4 instead. Furthermore, Piwil2 transfected cells exhibited a high tendency to form colony-like structures, partial staining for alkaline phosphatise (AP) activity, expression of germ layer markers and markers of the blood lineage. In terms of teratoma formation, Piwil2 transfected cells showed tumour outgrowth when injected subcutaneously in SCID mice. However, histological observations of the tumour sections revealed that tumours were not teratomas; instead, they were highly malignant tumours with rare signs of differentiation. This result is consistent with the suggestion that the Piwil2 gene is a cancer stem cell gene that is ectopically expressed in a variety of mammalian tumours. Although these results do not show full germ cell or i~S cell conversion of fibroblasts, the dramatic changes that were induced in the Piwil2 transfected cells, compared to the control cells, suggest this gene can act as a modulator of fundamental pathways such as proliferation and differentiation and bestow an unusual phenotype on fibroblasts. It is worth pointing out that some of the findings of this study, such as Oct4 and Terl19 expression in transfected fibroblasts, were not stable in spite of stable expression of the Piwil2 gene. The underlying reason for this might be some sort of dynamic effect of the Piwil2 gene on cell behaviour

The ability to reprogram adult somatic cells into induced pluripotent stem (iPS) cells could provide a valuable implement for in vitro disease modeling and drug discovery. More importantly, they may potentially serve as an unlimited source of cells for regenerative medicine. However, most of the iPS cells have been generated by retroviral vectors, and therefore they carry the risk of viral integration into the host genome. This problem prevents their use for clinical applications and regenerative medicine. mRNA-mediated delivery of reprogramming factors is an alternative approach for cellular reprogramming. mRNA-based reprogramming offers the advantage of being completely free of genomic integration and is therefore highly suitable for clinical translation. However, there are some limitations which must be overcome so that mRNA can be widely used for successful cellular reprogramming. In the current thesis, the attempt was to generate stable mRNA-iPS cells through overcoming those limitations. Several human donor cells were transfected with mRNA encoding reprogramming factors and the generation of two stable mRNA-iPS cell lines was shown. The resultant mRNA-iPS colonies were assessed for pluripotency markers. Their pluripotency features were evaluated by the viral-iPS cells produced by conventional retroviral vectors. It was noticed that the generation of mRNA-iPS cells was largely affected by the parental cells from which they were derived. However, characterization and evaluation of the generated mRNA-iPS cells proved their pluripotency states comparable to the viral-iPS cells. On the other hand, the aging hallmarks of the iPS cells were assessed in the second part of this thesis. The potential aging signatures of the iPS cells should be conducted before their use in clinical applications. Currently, there are controversial data regarding the ability of reprogramming to fully rejuvenate an aged somatic cell and reverse agerelated changes such as shortened telomeres, dysfunctional mitochondria and DNA damage. Moreover, mixed findings have been published regarding whether the iPS cells are fully rejuvenated or they might retain some of the aging hallmarks from the cells which they were derived. This thesis studied these controversies through the investigation of three hallmarks of aging including telomere length, mitochondrial alteration and DNA damage. Telomere elongation was indicated in the iPS cells. Furthermore, mitochondrial morphology and function were improved into more immature features in iPS cell lines than their corresponding fibroblasts. Moreover, the iPS cell lines were shown to have less amount of DNA damage compared to their parental fibroblasts. In summary, it can be concluded that generation of mRNA-iPS cells is largely affected by the primary donor cells from which they are derived. Furthermore, it seems that reprogramming enables reversion of aging signatures to a more youthful state.

The generation of induced pluripotent stem (iPS) cells is a useful tool for regenerative medicine. However, the risk of tumor development of the aforementioned cells should be addressed before they can be used for clinical applications. During the reprogramming process a number of signal pathways are activated, which may lead to direct differentiation of specific cell lineages prior to the cells reaching the pluripotent state. In order to test this hypothesis we designed a combined protocol of reprogramming and differentiation in an attempt to achieve direct differentiation of fibroblasts to specific cell lineages. Human fibroblasts were shortly reprogrammed by overexpression of four reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) and maintained in reprogramming media on a gelatin substrate for four days. These cells were defined as partially induced pluripotent stem (PiPS) cells. PiPS cells did not form tumours in vivo and differentiated into smooth muscle cells (SMCs) when seeded on a Collagen IV substrate and maintained in differentiation media (DM). The PiPS-SMCs expressed a panel of SMC markers such as SMA, SM22 and Calponin at mRNA and protein levels. Immunofluorescent staining of PiPS-SMCs showed positive staining for the above markers, demonstrating a typical SMC morphology. These cells displayed a greater potential to differentiate into SMCs than iPS cells. In order to elucidate the mechanism of PiPS cell differentiation into SMCs, data from a series of experiments indicated that the gene DKK3 was involved in SMC differentiation of PiPS cells. DKK3 was expressed in parallel with SMC markers, while its overexpression or stimulation induced SMC marker expression. Furthermore, DKK3 silencing resulted in downregulation of SMC markers on both the mRNA and protein levels. Finally, additional experiments revealed that the upregulation of SMC markers by DKK3 is mediated by activation of Wnt signalling through interaction of DKK3 with the transmembrane receptor Kremen 1. Therefore, we developed a protocol to generate SMCs from PiPS cells through a DKK3 signal pathway. These findings provide a new insight into the mechanisms of SMC differentiation with therapeutic potential to vascular disease.

Le cellule di Schwann (SC) sono cellule derivate dalla cresta neurale (NC) in grado di produrre la guaina mielinica avvolgendo gli assoni neuronali nel sistema nervoso periferico (PNS). I trapianti di SC potrebbero diventare un'opportunità terapeutica interessante per il trattamento delle lesioni del midollo spinale, dei nervi periferici e delle malattie demielinizzanti del PNS. Tuttavia, questi approcci terapeutici sono fortemente limitati dall'attuale mancanza di una fonte rinnovabile di SC. Le strategie di riprogrammazione cellulare si sono rivelate efficaci nel fornire una varietà di cellule specifiche per la modellizzazione delle malattie e per le procedure di trapianto di cellule, ottenute mediante sovraespressione di fattori di trascrizione cardine nello sviluppo embrionale del tipo cellulare di interesse. Ho identificato i due fattori di trascrizione Sox10 ed Egr2 in grado di generare cellule di Schwann indotte (iSCs) ad alta efficienza quando co-espresse in fibroblasti murini. Le iSC assomigliano a SC primarie nel profilo globale di espressione genica ed esprimono marcatori specifici di SC, tra cui S100ß, O4 e MPZ. Quando co-coltivati con espianti di gangli della radice dorsale di topo (DRG), le iSCs hanno generato guaine di mielina compatte organizzate in internodi Mbp+ intervallati da domini paranodali caspr + e domini nodali con canali del sodio. Al contrario, le iSC derivate da topi Twitcher hanno mostrato una grave perdita del potenziale mielinogenico, indicando le iSC come un sistema attraente per la modellazione in vitro delle malattie del PNS. Quindi, ho derivato iSCs da ratti sottoposti ad assotomia del nervo mediano seguito da trapianto di condotti di chitosano precedentemente seminati con iSC autologhe. Questi condotti con le iSC supportano e accelerano la rigenerazione dei nervi con un migliorato contenuto di mielina. Allo stesso modo, Sox10 ed Egr2 sono sufficienti per convertire i fibroblasti umani in iSC. Inoltre, la loro espressione facilita fortemente la differenziazione in SC delle cellule staminali pluripotenti indotte umane (iPSC), includendo nella strategia di riprogrammazione pochi passaggi intermedi che forniscono diversi stimoli trofici alle cellule differenzianti. In particolare, dopo la trasduzione lentivirale con i lentivirus esprimenti Sox10 ed Egr2, ho aggiunto piccole molecole neuralizzanti (SB431542 e LDN193189 in terreno iPSC), insieme a un terreno di differenziazione della cresta neurale (B27, acido ascorbico e FGF2 in terreno neurobasico) e infine un medium specifico per la crescita delle cellule di Schwann (Forskoline, NRG1, FGF2 in DMEM 10% FBS), fornendo una semplice procedura per ottenere un gran numero di SC omogenee e ben differenziate. Complessivamente, Sox10 ed Egr2 sono una combinazione unica di fattori per la generazione efficace di iSC mielinogeniche da fibroblasti di roditori e umani e da iPSC. Il processo rapido e diretto per generare iSC faciliterà la modellazione in vitro e gli approcci di trapianto di cellule autologhe per le malattie del PNS.<br>Schwann cells (SCs) are neural crest (NC) derived cells able to produce the myelin sheaths, wrapping neuronal axons in the peripheral nervous system (PNS). Transplantations of SCs might become an interesting therapeutic opportunity for the treatment of spinal cord and peripheral nerves injuries and demyelinating diseases of the PNS. However, these therapeutic approaches are strongly limited by the current lack of a renewable source of SCs. Cell reprogramming strategies have proven to be effective in providing a variety of tissue-specific cells for disease modelling, and cell transplantation procedure by over expression of cardinal developmental transcription factors of the interest cell type. I have identified the two transcription factors Sox10 and Egr2 able to generate induced Schwann Cells (iSCs) when co-expressed in murine fibroblasts with high efficiency. iSCs resembled primary SCs in global gene expression profiling and expressed cardinal markers of SCs including S100ß, O4 and MPZ. When co-cultured with mouse dorsal root ganglion (DRG) explants, iSCs generated compact myelin sheaths organized in Mbp+ internodes spaced by Caspr+ paranodal and Na+ channel nodal domains. Conversely, iSCs from Twitcher mice showed a severe loss in the myelinogenic potential, indicating iSCs as an attractive system for in vitro modeling of PNS diseases. Then, I derived iSCs from rats that were subjected to median nerve axotomy followed by transplantation of chitosan conduits previously seeded with autologous iSCs. These iSC-seeded conduits supported accelerated nerve regeneration with improved myelin content. Similarly, Sox10 and Egr2 are sufficient to convert human fibroblasts into iSCs. Moreover, their expression strongly facilitate the SC differentiation of human induced pluripotent stem cells (iPSCs), including in the reprogramming strategy few intermediate steps that provide different trophic stimuli to the differentiating cells. In particular, after the lentiviral transduction with the Sox10 and Egr2 expressing lentiviruses, I added neuralizing small molecules (SB431542 and LDN193189 in hiPS medium), together with a neural crest differentiation medium (B27, Ascorbic Acid and FGF2 in neurobasal medium), and finally a specific medium for Schwann cell growth (Forskoline, NRG1, FGF2 in DMEM 10% FBS), providing a simple procedure for obtaining a large number of homogeneous and well-differentiated SCs. Altogether, Sox10 and Egr2 is a unique combination of factors for the effective generation of myelinogenic iSCs from rodent as well as human fibroblasts and iPSCs. The fast and straightforward process to generate iSCs will facilitate in vitro disease modeling and autologous cell transplantation approaches for PNS diseases.

In this study, we assayed the capability of four genes implicated in embryonic specification of the cortico-cerebral field, Foxg1, Pax6, Emx2 and Lhx2, to reprogramm mouse embryonic fibroblasts toward neural identities. Lentivirus-mediated, TetON-dependent overexpression of Pax6 and Foxg1 transgenes specifically activated the neural stem cell (NSC) reporter Sox1-EGFP in a substantial fraction of engineered cells. The efficiency of this process was enhanced up to ten times by simultaneous inactivation of Trp53 and co-administration of a specific drug mix inhibiting HDACs, H3K27-HMTase and H3K4m2-demethylase. Remarkably, a fraction of the reprogrammed population expressed other NSC markers and retained its new identity, even upon transgenes switching off. When transferred into a pro-differentiative environment, Pax6/Foxg1-overexpressing cells activated the neuronal marker Tau-EGFP. Frequency of Tau-EGFP cells was almost doubled upon delayed delivery of Emx2 and Lhx2 transgenes. A further improvement of the neuron-like cells output was achieved by tonic inhibition of BMP and TGFb pathways. These Tau-EGFP cells showed a negative resting potential and displayed active electric responses, following injection of depolarizing currents.

Current usage of human embryonic stem cells (hES) and induced pluripotent stem cells (iPS) in clinical therapies and personalized medicine are limited as a result of ethical, technical and medical problems that arise from isolation and generation of these cells. Isolation of hES cells faces ethical problems associated with their derivation from human pre-implantation embryos. The most controversial aspect of hES cell isolation targets the generation of autologous hES cell lines which requires the transfer of a somatic-cell nucleus from the patient to an enucleated oocyte. While already established embryonic stem cell lines from IVF embryos can be used in a similar manner, lack of genetic identity can cause therapy rejection from the host, and prevent their use in personalized medicine. Induced pluripotent stem cells on the other hand, are generated from somatic cells that have been reprogrammed in vitro to behave like stem cells. While these cells can potentially be used for personalized medicine without the risk of rejection by the host system, derivation methods prevent their therapeutic use. The most efficient method used to generate iPS cells involves usage of viral particles which can result in viral DNA being integrated in the host cell’s genome and render these cells non-compliant for clinical therapies. Other methods not involving viral particles exist as well, but the reprogramming efficiency is too low and technical problems with generating large enough numbers of cells prevent these methods from being feasible approaches for clinical therapies. Direct reprogramming of a differentiated cell into a developmentally more plastic cell would offer alternatives to applications in regenerative medicine that currently depend on either embryonic stem cells (ES), adult stem cells or iPS cells. We hypothesize that Xenopus laevis egg cytoplasmic extract contains critical factors needed for reprogramming that may allow for non-viral, chemically defined derivation of human induced pluripotent/multipotent cells which can be maintained by addition of exogenous FGF2. In this thesis we investigated a new method for generation of multipotent cells through determining the ability of select fractions of Xenopus laevis egg extract to induce multipotency in already differentiated cells. We were able to identify select fractions from the extract that in combination with exogenously added FGF2 can reprogram and maintain the reprogrammed cells in an undifferentiated state. The findings of this work also determined that Xenopus laevis egg extract mRNA is required for achieving full reprogramming. The body of work presented in this thesis showed the ability of FGF2 isoforms to bind and activate select FGF receptor tyrosine kinases, act as extracellular mitogenic factors to support growth of hES cells in an undifferentiated state as well bind to nuclear DNA and affect expression of endogenous genes. Moreover, we showed that all FGF2 isoforms can induce expression of stem cell specific proteins in human dermal fibroblasts as well as extend lifespan of human dermal fibroblasts in vitro. In this work we identified HECW1, the gene coding for E3 ubiquitin ligase NEDL1, as a novel nuclear target for all FGF2 isoforms and showed that overexpression of recombinant FGF2 isoforms in human dermal fibroblasts can down regulate expression of HECW1 gene.

The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates. In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells. Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome.<br>Ph. D.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Orientador: Fernanda da Cruz Landim<br>Resumo: As pesquisas envolvendo a biologia das células-tronco abordam um amplo espectro de fenômenos, que vão desde o nível tecidual e celular, até o seu uso em terapias celulares. Esta crescente atenção sugere que é necessário estudar conceitos básicos da biologia das células-tronco para compreender completamente os processos de diferenciação funcional. Desta forma, o instrumento da reprogramação celular por meio da manipulação gênica fornece subsídios para melhor compreender os processos de renovação e diferenciação que constituem as características fundamentais das células-tronco. A obtenção dessas células em medicina veterinária visa validar diversos modelos experimentais domésticos, como o equino, na busca de novos fármacos e terapias alternativas para reabilitação. Uma série de estudos, porém, ainda são necessários para que tais aplicações sejam viáveis, uma vez que os mecanismos fundamentais das técnicas empregadas ainda não estão totalmente elucidados. Embora a reprogramação celular por meio de vetores virais tenha sido relatada com sucesso em diversas espécies animais, outras técnicas também podem ser empregadas, como o uso de transposons, sequências de DNAs capazes de se movimentar de uma região para outra no genoma de uma célula. Não se tem conhecimento de qual o melhor tipo celular a ser utilizado, e nem tão pouco qual a metodologia de reprogramação mais eficiente. Sabe-se que o cordão umbilical possui uma reserva rica em células-tronco mesenquimais, as quais por serem mu... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Researches on the biology of stem cells cover a broad spectrum of phenomena, ranging from tissue and cellular level, to their use in cell therapy. This growing attention suggests that is necessary to study basic concepts of stem cells organization in order to fully understand the functional differentiation processes. Thus, the cell reprogramming through gene manipulation provides grants to better understand the processes of renewal and differentiation which are the essential characteristics of stem cells. Obtaining these cells in veterinary medicine aims to validate various household experimental models, such as horses, on the search for new drugs and alternative therapies for rehabilitation. However, a number of studies is still necessary for such applications to be feasible, since the fundamental mechanisms of techniques employed are not fully elucidated yet. Although cell reprogramming using viral plasmid has been reported with success in several animal species, other techniques may also be employed, such use transposons, this is, DNAs sequences capable of moving from one region to another in the cell genome. The unawereness of what the best cell type to be used, and nor what is the most efficient reprogramming methodology. It is known that the cord has rich reserves mesenchymal stem cells, which are multipotent and can improve the efficiency of obtaining the induced Pluripotent Stem Cells (iPS) compared to the use of fibroblast, inefficient to be reprogrammed. The aim of this study was to obtain iPS through viral transfection and nonviral adult fibroblasts and equine cord cells, aiming to observe which transfection and cell type is more efficient for cell reprogramming. Both cell types was infected with viral vectors and transposons containing the genes OCT-4, SOX-2, c-MYC, and KLF-4; transformed cells were evaluated for morphology, immunocytochemistry... (Complete abstract click electronic access below)<br>Doutor

La terapia genica basata sull’utilizzo di vettori integranti è stata già applicata con successo per la cura di varie malattie genetiche come le malattie da accumulo lisosomiale (LSD), la beta-talassemia (β-Thal) e le immunodeficienze primarie (PID). L’immunodeficienza combinata grave legata al cromosoma X (SCID-X1) è una malattia monogenica letale causata da mutazioni del gene codificante la catena comune gamma del recettore per l’interleuchina 2 (IL2RG). I primi studi clinici per la SCID-X1 hanno mostrato il potenziale terapeutico della terapia genica basata su vettori integranti, risultando nella ricostituzione del compartimento linfoide grazie al vantaggio selettivo delle cellule geneticamente modificate. D’altra parte, tali studi hanno evidenziato il rischio di mutagenesi inserzionale dovuto all’integrazione casuale del virus nel genoma della cellula ospite e all’espressione non regolata del transgene, sottolineando la necessità di sviluppare nuove strategie di terapia genica più sicure. In questo lavoro, sfruttando la tecnologia delle Zinc-Finger Nucleasi (ZFN) per indurre una rottura del doppio filamento del DNA in maniera sito specifica e dei vettori lentivirali difettivi per l’integrazione (IDLV) per l’introduzione di un templato donatore, abbiamo impiegato il processo di riparazione del DNA guidata dall’omologia per la correzione delle mutazioni che causano la SCID-X1, ripristinando così la funzione genica e l’espressione fisiologica del gene IL2RG. Mediante l’integrazione di un cDNA correttivo del gene IL2RG a valle del promotore endogeno sia in cellule B linfoblastodi, che esprimono costitutivamente la catena gamma comune, sia in linfociti T da donatori sani, che richiedono IL2RG per la loro sopravvivenza, abbiamo dimostrato la funzionalità e l’attività fisiologica del gene modificato. Abbiamo quindi accoppiato la correzione genica con la selezione delle cellule mediante l’inclusione di una cassetta excidibile di espressione della GFP o della resistenza alla puromicina (PuroR) a valle del cDNA correttivo, al fine di correggere fibroblasti, che normalmente non esprimono IL2R, derivati da pazienti SCID-X1. Abbiamo quindi ottenuto una popolazione di fibroblasti corretti che abbiamo “ riprogrammato” mediante un nuovo vettore di reprogramming che esprime i fattori di trascrizione (SOX2, OCT4, KLf4) e il microRNA cluster 367, generando così una fonte illimitata di cellule staminali pluripotenti indotte (iPSC) geneticamente corrette di interesse terapeutico. L’espressione transiente della Cre-ricombinasi mediante IDLV ha inoltre permesso l’excisione del vettore di reprogramming e della cassetta di selezione, permettendo così l’ottenimento di cellule iPSC corrette, prive di vettore e con un normale cariotipo. Infine, attraverso il differenziamento delle cellule iPSC in progenitori T-linfoidi, un tipo cellulare assente nei pazienti SCID-X1, e l’osservazione di un vantaggio selettivo delle cellule linfoidi derivate dalle iPSC corrette, abbiamo dimostrato la correzione funzionale dell’allele IL2RG mutato. In conclusione questi dati dimostrano la validità della nostra strategia di integrazione sito-specifica che, mediante la correzione e la riprogrammazione cellulare, consente di ottenere cellule iPSC geneticamente corrette, aprendo la strada a nuove opportunità terapeutiche più sicure per il trattamento della SCID-X1.<br>Gene replacement by integrating vectors has been successfully used to treat several inherited diseases, such as Lysosomal Storage Disorders (LSD), Thalassemia and Primary Immunodeficiencies (PIDs). X-linked Combined Immunodeficiency (SCID-X1) is a fatal monogenic disorder, caused by mutation of the Interleukin 2 Receptor common γ-chain (IL2RG) gene. For SCID-X1, the early clinical studies have clearly shown the therapeutic potential of integrating vector based gene replacement therapy, which achieved efficient lymphoid reconstitution thanks to the selective growth advantage of the genetically modified cells. However, these studies also highlighted the potential risk of insertional mutagenesis due to random integration of the vector into the host cell genome and to unregulated transgene expression, thus calling for the development of safer gene therapy approaches. Here, by combining the Zinc Finger Nuclease (ZFNs) technology to induce site-specific DNA double-strand breaks (DSB) and of Integrase-Defective Lentiviral Vector (IDLV) to deliver a corrective donor template, we exploited Homology Driven Repair (HDR) to correct SCID-X1 mutation in situ, restoring both physiological expression and function of the IL2RG gene . By knocking-in a corrective IL2RG cDNA transgene downstream of its endogenous promoter in B-lymphoblastoid cells, which constitutively express IL2RG, and in primary T-lymphocytes, which requires IL2RG for their survival and growth, we provide evidence of physiologic activity of the gene-edited IL2RG gene. By including an excisable GFP- or a Puromycin Resistance (PuroR) expression cassette downstream of the corrective cDNA, we coupled correction with exogenous selection of corrected SCID-X1 primary fibroblasts, which do not physiologically express IL2RG, and obtained an enriched population of gene-corrected cells. We then reverted this population to pluripotency by using a novel reprogramming vector that expresses OCT4, SOX2, KLF4 and microRNA cluster 302-367 to obtain a potentially unlimited source of gene-corrected induced pluripotent stem cells (iPSC). We thus generated several gene-corrected bona-fide iPSCs, as confirmed by molecular analyses for targeted integration, which were characterized for their pluripotent state. IDLV-mediated transient delivery of the Cre-recombinase resulted in the co-excision of the reprogramming vector together with the selector cassette, thus allowing the generation of several gene-corrected, reprogramming-factor free iPSCs with normal karyotypes. Finally, by differentiating corrected iPSC to T-lymphoid progenitor cells, which are lacking in SCID-X1 patients, and showing a selective growth advantage of those derived from corrected iPSCs, we provide evidence of the functional correction of the IL2RG mutant allele. Overall these data demonstrate the feasibility of our targeted gene editing strategy, which couples gene correction with cell reprogramming to generate disease-free IPSC, thus paving the way for the development of novel and safer therapeutic approaches for SCID-X1.

Induced pluripotent stem (iPS) cells have the potential to generate a wide array of cell types from multiple lineages that enable us to explore the mechanisms that are involved in the conversion of cell states. The reprogramming process that generates iPS cells is complex, but since its discovery, technical advancements and improvements in the methodology have improved the speed and efficiency of generating integration-free, clinically relevant iPS cells. However, despite improvements, the mechanisms of reprogramming are not fully understood and so the process remains largely inefficient and slow. It has been reported that reprogramming mediated through the delivery of exogenous mRNAs encoding OCT4, SOX2, KLF4 and cMYC is a fast and efficient method for generating integration-free iPS cells. Here we show that mRNA reprogramming can be enhanced further by employing an mRNA dose-ramping approach that provides greater control of the dose of mRNA that is introduced into the target cells. This improvement upon existing methods promotes the viability of the target cells during reprogramming which in turn improves the efficiency, speed and success of generating iPS cells. We also show that an optimisation to the reprogramming factor cocktail, replacing OCT4 with a fusion between OCT4 and the transcriptional activation domain of MYOD1 – called M3O, further improves the kinetics of reprogramming. Reprogramming disease cells is also possible in that several iPS cell-disease models have been established that have successfully modelled aspects of disease development in vitro. Here we show the applicability of using the mRNA approach we have developed, on neuroblastoma cells and the characterisation of iPS cells reprogrammed from neuroblastoma cells using OCT4, SOX2, KLF4 and cMYC delivered using Sendai viral vectors. Finally, we demonstrate how human fibroblasts introduced to a vector encoding MYOD1 causes them to transdifferentiate in to myoblast-like cells without a genomic footprint. Together this data demonstrates how integration-free mRNA can be used to control gene expression to direct cell fate through reprogramming and transdifferentiation. This mRNA approach provides proof of concept that warrants the testing of other genes to explore their function in reprogramming and other pathways that govern cell fate.

Somatic cells could be reprogrammed into an ES-like state called induced pluripotent stem cells (iPSCs) by expression of four transcriptional factors: Oct4, Sox2, Klf4 and cMyc. iPSCs have full potentials to generate cells of all lineages and have become a valuable tool to understand human development and disease pathogenesis. However, reprogramming process suffers from extremely low efficiency and the molecular mechanism remains poorly understood. This dissertation is focused on studying the role of small non-coding RNAs (microRNAs) and kinases during the reprogramming process in order to understand how it is regulated and why only a small percentage of cells could achieve fully reprogrammed state. We demonstrate that loss of microRNA biogenesis pathway abolished the potential of mouse embryonic fibroblasts (MEFs) to be reprogrammed and revealed that several clusters of mES-specific microRNAs were highly induced by four factors during early stage of reprogramming. Among them, miR-93 and 106b were further confirmed to enhance iPSC generation by promoting mesenchymal-to-epithelial transition (MET) and targeting key p53 and TGFβ pathway components: p21 and Tgfbr2, which are important barrier genes to the process. To expand our view of microRNAs function during reprogramming, a systematic approach was used to analyze microRNA expression profile in iPSC-enriched early cell population. From a list of candiate microRNAs, miR-135b was found to be most highly induced and promoted reprogramming. Subsequent analysis revealed that it targeted an extracellular matrix network by directly modulating key regulator Wisp1. By regulating several downstream ECM genes including Tgfbi, Nov, Dkk2 and Igfbp5, Wisp1 coordinated IGF, TGFβ and Wnt signaling pathways, all of which were strongly involved in the reprogramming process. Therefore, we have identified a microRNA-regulated network that modulates somatic cell reprogramming, involving both intracellular and extracellular networks. In addition to microRNAs, in order to identify new regulators and signaling pathways of reprogramming, we utilized small molecule kinase inhibitors. A collection of 244 kinase inhibitors were screened for both enhancers and inhibitors of the process. We identified that inhibition of several novel kinases including p38, IP3K and Aurora kinase could significantly enhance iPSC generation, the effects of which were also confirmed by RNAi of specific target genes. Further characterization revealed that inhibition of Aurora A kinase enhanced phosphorylation and inactivation of GSK3β, a process mediated by Akt kinase. All together, in this dissertation, we have identified novel role of both small non-coding RNAs and kinases in regulating the reprogramming of MEFs to iPSCs.

La reprogrammation, qui est la réversion d’un noyau d’un état somatique vers un état moins différencié, constitue un enjeu majeur pour la thérapie cellulaire. Cependant, les mécanismes initiaux qui président à la reprogrammation restent mal connus. Le transfert nucléaire (clonage) met à profit les propriétés de reprogrammation uniques du cytoplasme ovocytaire, et constitue une approche expérimentale intéressante pour analyser ces processus. Le but de cette thèse est d’étudier la différence de capacité de cellules fibroblastiques à être reprogrammées efficacement, en tirant partie d’une situation-modèle d'efficacité différentielle de reprogrammation après clonage chez le bovin. Ce modèle est constitué de deux lots de fibroblastes donneurs de noyaux, qui forment des embryons clonés dont la différence d’efficacité de développement à terme varie d’un facteur 8. L’analyse des cellules donneuses a montré une augmentation des anomalies de ploïdie dans les cellules à faible potentiel, et la similitude transcriptomique entre les cellules donneuses, alors que la comparaison des transcriptomes des embryonsclonés a montré des différences de reprogrammation de l’expression génique dès le stade suivant l’activation du génome embryonnaire. Des différences de méthylation de l’ADN entre cellules donneuses ont été observées dans les promoteurs de gènes candidats différentiellement reprogrammés, ainsi que dans une analyse plus globale par RRBS. Nous avons enfin étudié la distribution des cellules filles des deux premiers blastomères au stade blastocyste, la distribution « orthogonale » et l’aptitude au développement à terme des embryons de souris clonés étant liées (Liu et al., 2012). Nous avons montré l’existence de trois distributions dans les embryons fécondés mais n’avons pas observé de différence de proportions de celles-ci entre embryons bovins clonés. En conclusion, dans notre modèle, la distribution des cellules filles des deux premiers blastomères au stade blastocyste ne semble pas associée à l’efficacité de reprogrammation dans les embryons bovins clonés, contrairement aux différences épigénétiques entre cellules donneuses<br>Reprogramming, which is the return of a nucleus from a somatic state to a less differentiated state, is a major issue for cell therapy. However, the initial mechanisms governing the reprogramming remain poorly understood. Nuclear transfer (cloning) takes advantage of the unique reprogramming properties of the oocyte cytoplasm, and therefore is an interesting experimental approach to analyze these processes. The aim of this thesis is to study the difference in fibroblasts’ ability to be reprogrammed by taking advantage of a model-situation of differential reprogramming efficiency after cloning in cattle. This model consists of two batches of donor fibroblasts, which form cloned embryos having an 8 fold difference in development to term efficiency. Analysis of donor cells has shown increase ploidy abnormalities in cells of low potential, and transcriptomic similarity between the donor cells, whereas comparison ofcloned embryos transcriptomes showed gene expression reprogramming differences just after embryonic genome activation. Differences in DNA methylation between donor cells were observed in the promoters of candidate genes differentially reprogrammed and in a more comprehensive analysis by RRBS. Finally we studied the distribution of the first two blastomeres’ daughter cells at the blastocyst stage, as an "orthogonal" distribution and development to term of mice cloned embryos are linked (Liu et al., 2012). We have shown the existence of three distributions in the fertilized embryos but haven’t seen any difference of proportions between bovine cloned embryos. In conclusion, in our model, the distribution of the first two blastomeres’ daughter cells at the blastocyst stage does not seem related to the reprogramming efficiency in bovine cloned embryos, unlike epigenetic differences between donor cells

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014<br>Células diferenciadas podem ser reprogramadas de modo a voltarem a ser pluripotentes e se transformarem em outros tipos de células através da inserção de factores específicos. No entanto a reprogramação de células somáticas de modo a se obter células estaminais pluripotentes induzidas (iPS), tem um tempo de reprogramação é relativamente longo, a eficiência da reprogramação ainda é relativamente baixa, alguns dos factores de pluripotencia que são usados para este processo são pro-oncogenes, e ainda existe a dificuldade de ultrapassar a senescência. Através de células embrionárias fibroblasticas de ratinho (MEF’s) o objectivo é reprogramar com os 4 factores de transcrição idenficados como genes de pluripotencia, Sox2, Nanog, Klf4 e c-Myc, acrescentando o mais um factor de transcrição Cited2 e usando estas células nas passagens P2 e P4. O Cited 2 é um regulador de transcrição de genes envolvido em muitas cadeias de transcrição biológicas, sendo esta capaz de induzir a proliferação das células quer de ratinhos como de humanos, é considerado um “anti-senescente”, tem a capacidade de activar a transcrição dos genes envolvidos na pluripotencia. Para este trabalho foram criados vectores virais, dois retrovírus, sendo um deles o Cited2 de modo a ser sobreexpresso nas células contendo GFP acoplada (KRK1), outro apenas com GFP que servirá de controlo (LZRS) e dois lentivírus que permitiram a inserção e activação da transcrição dos 4 factores de Yamanaka (respectivamente TET STENCCA e M2RTTA). Serão analisadas morfologias das colonias obtidas através da reprogramação, serão feitos qRT-PCR às colonias de modo a se perceber se expressão os factores de pluripotência das células estaminais embrionárias (ESC), e se através da formação de corpos embrionários (EB’s) através das iPS obtidas estas são capazes de dar origem às três linhas germinativas, mesoderme, ectoderme e endoderme, este processo será realizado por qRT-PCR. Após análise dos resultados obtidos, pode-se concluir, que o Cited2 nas MEF’s P2 possibilita o aparecimento precoce dos agregados de colónias em relação às MEF’s que não têm Cited2 exógeno, enquanto nas MEF’s P4 com Cited2 sobreexpresso, consegue-se ultrapassar a senescência e obter-se iPS com valores bastante semelhantes entre si a nível da transcrição dos factores de pluripotencia (Nanog, Sox2, Oct4 e Rex1).

The ability to reprogram adult somatic cells into induced pluripotent stem (iPS) cells could provide a valuable implement for in vitro disease modeling and drug discovery. More importantly, they may potentially serve as an unlimited source of cells for regenerative medicine. However, most of the iPS cells have been generated by retroviral vectors, and therefore they carry the risk of viral integration into the host genome. This problem prevents their use for clinical applications and regenerative medicine. mRNA-mediated delivery of reprogramming factors is an alternative approach for cellular reprogramming. mRNA-based reprogramming offers the advantage of being completely free of genomic integration and is therefore highly suitable for clinical translation. However, there are some limitations which must be overcome so that mRNA can be widely used for successful cellular reprogramming. In the current thesis, the attempt was to generate stable mRNA-iPS cells through overcoming those limitations. Several human donor cells were transfected with mRNA encoding reprogramming factors and the generation of two stable mRNA-iPS cell lines was shown. The resultant mRNA-iPS colonies were assessed for pluripotency markers. Their pluripotency features were evaluated by the viral-iPS cells produced by conventional retroviral vectors. It was noticed that the generation of mRNA-iPS cells was largely affected by the parental cells from which they were derived. However, characterization and evaluation of the generated mRNA-iPS cells proved their pluripotency states comparable to the viral-iPS cells. On the other hand, the aging hallmarks of the iPS cells were assessed in the second part of this thesis. The potential aging signatures of the iPS cells should be conducted before their use in clinical applications. Currently, there are controversial data regarding the ability of reprogramming to fully rejuvenate an aged somatic cell and reverse agerelated changes such as shortened telomeres, dysfunctional mitochondria and DNA damage. Moreover, mixed findings have been published regarding whether the iPS cells are fully rejuvenated or they might retain some of the aging hallmarks from the cells which they were derived. This thesis studied these controversies through the investigation of three hallmarks of aging including telomere length, mitochondrial alteration and DNA damage. Telomere elongation was indicated in the iPS cells. Furthermore, mitochondrial morphology and function were improved into more immature features in iPS cell lines than their corresponding fibroblasts. Moreover, the iPS cell lines were shown to have less amount of DNA damage compared to their parental fibroblasts. In summary, it can be concluded that generation of mRNA-iPS cells is largely affected by the primary donor cells from which they are derived. Furthermore, it seems that reprogramming enables reversion of aging signatures to a more youthful state.

ROSA, Fábio Alexandre Fiúza - Direct Reprogramming of Fibroblasts to Dendritic Cells for Immunotherapy. Coimbra : [s.n.], 2016. Dissertação de Mestrado.<br>The maintenance of cellular identity relies on stable and complex gene regulatory networks. However, several studies have shown that cell fate can be reversed or modified by defined sets of lineage specific Transcription Factors (TFs). The process of direct cellular reprogramming holds promise for the generation of autologous cells for regenerative medicine. In the immunotherapy field, the use of immune modulatory cells enables the manipulation of patients ‘own immune system to target cancer cells. In this context we aim to apply direct cellular reprogramming for the generation of Dendritic Cells (DCs) as ideal antigen-presenting cells to kick-start adaptive immune responses. Here, TF-mediated direct reprogramming approach was established to generate DCs from fibroblasts. First, we employ a combination of literature mining and computational analysis to identify candidate TFs to induce DC fate in vitro. Candidate TFs were selectively expressed in DC populations in both mice and humans and their disruption caused abnormal adaptive immune phenotypes in mice. This analysis generated 19 candidate TFs with key developmental roles in the DC lineage. We have expressed a set of these TFs using a reprogramming proven Doxycycline-inducible lentivirus in mouse embryonic fibroblasts (MEFs). Employing transgenic MEFs harbouring the DC-specific reporter Clec9a-Cre X R26-stop-Tomato, a minimal combination of 4 TFs was identified. This set of 4 TFs activated the DC-specific reporter and generated tdTomato+ cells. TdTomato+ cells acquired DC-like morphology with increased size and complexity. Moreover, a percentage of tdTomato+ cells expressed Major Histocompatibility Complex (MHC) Class II at the cell surface, a critical molecule for antigen presenting function. Finally, overexpression of the 4TFs in Human Dermal Fibroblasts generated cells with DC-like morphology. These morphological changes emerged with similar timing and efficiency in mouse and Human, supporting species conservation of transcriptional regulators underlying DC commitment. Collectively, DC-like cells were generated via a TF-mediated direct reprogramming approach. The results presented in this study highlight the potential of direct reprogramming to a better understanding of transcriptional events underlying lineage specification and to generate immune modulatory cells for immunotherapy.

碩士<br>國立臺灣大學<br>臨床牙醫學研究所<br>101<br>Induced pluripotent stem cell (iPS), generated from somatic cells by the four transcription factors, has been startling development of reprogramming technology. Since the integration of vectors is not fully predictable, a flood of studies have been publishing, including protein based-DNA free method. This method required abundant protein extracts, which were often collected by embryonic stem cells or teratocarcinoma, were harder to be applied clinically. Human cord blood has been established as a stable and potential source of mesenchymal stromal cells (cbMSC). Human gingival fibroblasts (GF) characterize as high proliferation rate and friendly collected by dentists. Although GF has recently been proved as a potential source by the traditional four-factor reprogramming via retrovirus, both cbMSC and GF have never been used in any DNA free reprogramming methods. Streptolysin O (SLO), a pore forming endotoxin, is widely used in protein delivery for DNA free reprograming. However, the response of GF treated with low sublytic dose has never been studied as well. Thus, we hypothesize that GF could be reprogrammed by protein extracs, and the research goal of the study is to reprogram GF by cbMSC’s protein extracts. Materials and methods: The questions remained were evaluated as follows: SLO caused cell viability change was evaluated by MTT assay, the membrane permeabiliting efficiency was evaluated by fluorescence stain, and phosphorylation of signal pathways were evaluated by Western Blot. After adding stem cell extracts, the reprogramming effect was examined by RT-PCR and DNA microarray for mRNA level. The differentiation ability was also checked as well. Results: Though SLO showed little effect at the concentration below 1000ng/mL, it caused membrane permeabilization in both GF and SF. Phosphorylation of ERK1/2 and P38 were also induced when SLO was treated. Adding cbMSC extracts in SLO treated GF increased the mRNA expression of OCT4 and NANOG, which were regarded as stem cell markers. Furthermore, these treated cells differentiated better in adipogenic, osteogenic, and neurogenic induction. In conclusion, cbMSC extracts could reprogram SLO treated GF causing changes in genetic level and differentiation ability.

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina<br>As células estaminais hematopoiéticas (HSCs) são capazes de se auto-renovar e diferenciar em todos os tipos de células sanguíneas. Estas características fazem com que a transplantação de HSCs seja o principal tratamento contra doenças hematológicas. A incompatibilidade entre dador-paciente e o número insuficiente de HSCs que são obtidas para transplantação limitam o sucesso deste tipo de terapia celular. De forma a ultrapassar estas limitações, a expansão destas células in vitro seria a solução, mas este processo continua limitado e as HSCs acabam por perder a capacidade de se auto-renovarem. A reprogramação directa de células somáticas, mediada por factores de transcrição, abriu novas portas na área da medicina regenerativa de forma a HSCT mais personalizados. Apesar da baixa eficiência, a sobre-expressão dos factores de transcrição Gata2, Gfi1b e cFos permitiu a conversão de fibroblastos em HSCs estabelecendo um novo método de geração de HSCs indicadas para o paciente. Assim, uma melhor compreensão de como o complexo de reprogramação hematopoiética funciona e as interacções entre estes três factores de transcrição com outros componentes do complexo pode fornecer informação importante que permita o aumento da eficiência deste processo de forma a gerar HSCs com maior potencial de transplantação. Neste trabalho, defini os domínios do Gata2 necessários para interagir com o Gfi1b, cFos e com a LSD1/CoREST1 e mais importante, o papel catalítico do complexo LSD1/CoREST durante a reprogramação. Gata2 regula a expressão do gene Kdm1a, co-immunoprecipitações identificaram os domínios necessários para a interacção do Gata2 com os outros dois factores de transcrição, Gfi1b e cFos, mas também com a LSD1/CoREST1. O domínio que contem a sequência de localização nuclear (NLS) do Gata2 parece essencial na interacção com o Gfi1b, mas também com a LSD1/CoREST1, no núcleo. Os zinc fingers localizados no C-terminal e no N-terminal do Gata2 mostraram-se importantes na interação com a LSD1/CoREST1 e com o cFos, respectivamente. cFos também interage com o domínio transactivador do Gata2 no C-terminal, realçando as múltiplas vias necessárias à formação do complexo hemogénico. Esta cooperação entre os factores de transcrição e a LSD1/CoREST1 levanta a hipótese que este complexo pode ser essencial na reprogramação hematopoiética. LSD1 foi inibida farmacologicamente durante a reprogramação de fibroblastos em HSCs. A inibição com dois inibidores estruturalmente diferentes levou à diminuição drástica do processo de reprogramação demostrando que a LSD1 tem um importante papel catalítico durante este processo. Em suma, este estudo identifica as interacções funcionais entre o Gata2, Gfi1b, cFos e a LSD1/CoREST1 e o papel vital deste regulador epigenético durante a reprogramação hematopoiética e aquisição de HSCs. Mais informação sobre a regulação deste complexo hematopoiético pode aumentar a eficiência do processo aproximando esta tecnologia da translação para a clínica.<br>Hematopoietic stem cells (HSCs) are able of self-renewal and differentiation into all blood cell lineages. Due to this ability, hematopoietic stem cell transplantation (HSCT) constitutes treatment for a diversity of hematological disorders. Incompatibility between donor and host and the insufficient number of HSCs obtained for transplantation have limited the success of this cellular therapy. To overcome these limitations, expansion of HSCs in vitro has been explored, but this process is a changeling process as HSCs quickly lose stem cell properties upon expansion. Direct reprogramming mediated by transcription factors (TFs) of somatic cells is opening new routes for regenerative medicine and personalized HSCT. Albeit at low efficiency, combined expression of Gata2, Gfi1b and cFos induces reprogramming of fibroblasts into HSC-like cells providing a novel alternative to generate patient-specific HSCs. A better understanding of hemogenic reprogramming and the interactions between these three TFs with each other and with other players will provide valuable information to increase the efficiency of the process and to generate transplantable HSCs that can be used in the clinic. Here, I have defined Gata2 protein domains required to interact with Gfi1b, cFos and LSD1/CoREST1. Gata2 regulates the expression of the Kdm1a gene that encodes LSD1 and co-immunoprecipitations experiments revealed multiple domains required for the interaction of Gata2 with cFos, Gfi1b and LSD1/CoREST1. The nuclear localization sequence of Gata2 is essential for the interaction with Gfi1b but also with LSD1/CoREST1 in the nucleus. C-terminal zinc finger and the N-terminal zinc finger of Gata2 are important for the interaction with LSD1/CoREST1 and cFos, respectively. cFos also interacts with the C-transactivator domain of Gata2, highlighting multiple regulatory pathways involved in the establishment of this “hemogenic complex”. Given this cooperation between TFs and LSD1/CoREST1 I hypothesize that this complex may be essential for the hemogenic reprogramming. LSD1 was pharmacological inhibited during hematopoietic reprogramming into HSC-like cells. Inhibition with two structurally unrelated small molecules led to a drastic decrease of reprogramming efficiency implicating the catalytic function of LSD1/CoREST complex during the hemogenic reprogramming. Overall, this study identified functional interactions between of Gata2, Gfi1b, cFos and LSD1/CoREST1 and the vital role of this epigenetic regulator during hematopoietic reprogramming and acquisition of the HSC fate. This study paves the way for the regulation of this hemogenic complex bringing high-efficiency hemogenic reprogramming one step closer to clinical translation.<br>Outro - O projecto foi financiado pela Fundação para a Ciência e a Tecnologia, referência PTDC/BIM-MED/0075/2014 designado “Mecanismos da indução hemogénica em fibroblastos humanos”. NOTA: Esta informação foi colocada nesta secção porque o projecto em causa não constava na lista cedida.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2013<br>Hepatitis C is a liver disease that results from an infection by the Hepatitis C virus. It can range between severe to moderate illness, with variable time. Today, the most infected people are associate with drugs and injectable material. Until 1992, an individual was mostly infected by blood transfusions or organ transplants. This work reflects, therefore, the attempt to develop different strategies to the detailed study of hepatitis C virus, using a novel cellular strategy. The aim of the study is to obtain the iPSCs from fibroblasts by infected patients with the virus. Subsequently, it will be made differentiate into hepatocytes like cells. This development happens through progressive steps of cellular manipulation with many different conditions, culture mediums and techniques. Then, these cells are used to viral broader study, with results that mimic liver human conditions. In parallel, it was also tested forty five plasma samples from infected patients to comparing infection levels of different HCV genotypes as well as the response to drug therapy. The results were satisfactory once it was accomplished iPSC production, and it was possible to obtain conclusions with the RNA analysis.<br>A hepatite c é uma doença hepática resultante da infeção provocada pelo vírus da hepatite c. A doença pode variar de severa a ligeira, com duração diversa. Atualmente, a maioria dos indivíduos infetados estão associados ao contacto com drogas e material injetável. Até 1992, um indivíduo era maioritariamente infetado através de transfusões sanguíneas ou transplantes de órgãos. Este trabalho, reflete, por isso, a tentativa de desenvolver diferentes estratégias para o estudo pormenorizado do vírus da Hepatite C, recorrendo a uma estratégia celular inovadora. O estudo pretende obter o desenvolvimento de células iPSC a partir de fibroblastos de pacientes infetados com o vírus. Posteriormente será efetuada a sua diferenciação em hepatocytes like cells. Este desenvolvimento dá-se através de etapas progressivas de manipulação celular, com auxílio de diversas condições, meios de cultura e técnicas. Estas células são depois usadas para um estudo viral mais amplo, com resultados que mimetizam as condições hepáticas humanas. Em paralelo foi também testado o plasma de quarenta e cinco doentes infetados, com o objetivo de comparar níveis de infeção a partir de diferentes genótipos de HCV, assim como a resposta ao tratamento farmacológico. Os resultados foram satisfatórios uma vez ter sido conseguida a obtenção de iPSC, assim como foi possível tirar conclusões em relação às análises de RNA efetuadas.<br>Project developed under ERASMUS Research training program, at CBH – Centre de Biologie Humane, Hôpital Sud, Université de Picardie Jules Verne. Faculté de Pharmacie. Amiens – France.

碩士<br>國立臺灣大學<br>高分子科學與工程學研究所<br>107<br>Fullerenes have the unique biocompatibility and photoelectric properties and are candidate materials for biomedical applications. Several cell membrane proteins in nature such as bacteriorhodopsin also have photoelectric properties. Highly expressible bacteriorhodopsin (HEBR) is a novel light-sensitive opsin that has the potential to trigger neural activities through optogenetic modulation. In this study, we delivered HEBR plasmids to human fibroblasts and exposed the cells to C60 fullerene self-assembled two-dimensional nanosheets. Results showed that the above approach combined with light stimulation (3 second duration and three times per day) may promote the reprogramming and differentiation of human fibroblasts into neural-like cells in 7 days without any neural induction medium. The special photoelectric properties of fullerenes as culture substrates and transfected HEBR on cell membrane may provide a new optogenetic platform for regulating the location (C60 nanosheet) and time (frequency of light illumination) for human fibroblasts to become neural-like cells, and may be applied to improve neural regeneration in the future.

Heart disease is one of the leading causes of mortality in developed countries. The associated pathology is typically characterized by the loss of cardiomyocytes that leads, eventually, to heart failure. Presently, there are many promising strategies for cardiac regeneration. Direct cardiac reprogramming is becoming known as a novel therapeutic approach to regenerate injured hearts. Direct cardiac reprogramming is a simple and quick process however, the molecular mechanisms of cardiac reprogramming and cardiomyocyte-like cells functional maturation remain to be understood. Direct cardiac reprogramming has great potential to become one of the main strategies for regenerative medicine in heart failure since fibroblasts, contrary to cardiomyocytes which do not divide, are easily available in the heart, they are a large population of cells in the heart, which become activated and turn to myofibroblasts, contributing to fibrosis after cardiac injury. Currently it is known that a specific combination of three transcription factors, Mef2c, Gata4 and Tbx5 (MGT), are enough to reprogram non-myocyte mouse heart cells into induced cardiomyocyte-like cells. Nevertheless, human fibroblasts when infected with MGT appeared to have a small percentage of conversion. With MGT retrovirus we successfully transfected: mouse adult fibroblasts (MAFs), Feeders and Gm 03348 (human fibroblasts with 10 years old). Through qPCR analysis we evaluated the expression of lncRNAs: Gm 15856, Mir22hg, Gm 027028 and Gm 28592. Our goal was to understand which lncRNAs are the best candidates to knockdown in order no enhance direct cardiac reprogramming. In addition, we studied how nutrient manipulation in cell culture media can influence direct cardiac reprogramming. It was found that media with higher levels of glucose and glutamine had larger rates of cellular survival and proliferation.<br>As doenças cardíacas são uma das principais causas de mortalidade nos países desenvolvidos. A patologia associada é tipicamente caracterizada pela perda de cardiomiócitos que leva, eventualmente, à insuficiência cardíaca. Atualmente, existem muitas estratégias promissoras para a regeneração cardíaca. A reprogramação cardíaca direta tem se tornado conhecida como uma nova abordagem terapêutica para regeneração cardíaca depois de uma lesão. A reprogramação cardíaca direta é um processo simples e rápido, no entanto os seus mecanismos moleculares e de maturação celular continuam maioritariamente desconhecidos. A reprogramação cardíaca direta é uma abordagem terapêutica com grande potencial para se tornar uma das principais estratégias da medicina regenerativa no combate à insuficiência cardíaca, uma vez que os fibroblastos estão facilmente disponíveis no coração e dividem-se facilmente ao contrário dos cardiomiócitos. Os fibroblastos cardíacos são uma população alargada no coração que, após uma lesão, tornam-se em miofibroblastos ativos contribuindo para a fibrose. Atualmente, sabe-se que uma combinação específica de três fatores de transcrição, Mef2c, Gata4 e Tbx5 (MGT), é suficiente para reprogramar fibroblastos cardíacos de ratinho em cardiomiócitos induzidos. Por outro lado, quando fibroblastos humanos são infetados com MGT apresentam uma pequena percentagem de conversão. Com o retrovírus MGT transfectamos com sucesso: fibroblastos adultos de ratinho (MAFs), Feeders e Gm 03348 (fibroblastos humanos com 10 anos de idade). Através da análise de qPCR, avaliamos a expressão dos lncRNAs: Gm 15856, Mir22hg, Gm 027028 e Gm 28592. O nosso objetivo foi estudar quais os lncRNAs são os melhores candidatos para knockdown, e assim melhorar a eficiência da reprogramação cardíaca direta. Para além disso, estudamos como a manipulação de nutrientes nos meios de cultura pode influenciar a reprogramação cardíaca direta. Verificou-se que meios com níveis mais altos de glucose e glutamina apresentaram maiores taxas de sobrevivência e proliferação celular.<br>Mestrado em Biologia Molecular e Celular

碩士<br>國立臺灣大學<br>高分子科學與工程學研究所<br>106<br>3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermoresponsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ~190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration.

<p>The recent discovery that fibroblasts can be reprogrammed directly to functional neurons with lentivirus has reinvigorated the belief that autologous human cell therapies against many neurodegenerative diseases may be achievable in the near future. To increase the clinical translatability of this approach, we have developed a technique to perform this direct conversion without the use of virus. We transfected fibroblasts with plasmids condensed into non-viral nanoparticulate carriers with a bioerodible peptidomimetic polymer, pCBA-ABOL. Gene delivery with pCBA-ABOL was exceptionally effective and nontoxic, producing high transfection efficiencies and enabling serial dosing of plasmid cocktails. We delivered plasmids encoding neural lineage-instructive transcription factors to primary mouse embryonic fibroblasts (PMEFs), eliciting: drastic morphological changes, activation of endogenous neuronal transcription factor expression, neuronal promoter activity, and the appearance of neuronal proteins. Serial dosing of pCBA-ABOL complexes produced increasingly higher yields of these non-virally induced neurons (NiNs). The majority of NiNs fired action potentials under patch clamp. This is the first description of a method capable of directly inducing functional neuronal cells from fibroblasts without the use of virus.</p><p>We then moved on to further increase the yield of NiN generation, in an effort to produce a sufficient quantity of neurons for cell therapies. Informed by neurodevelopmental cues and by precedents set by the induced pluripotent stem cell (iPSC) field, we performed non-viral neuronal reprogramming in the presence of soluble microenvironmental factors that either inhibited GSK-3beta; and SMAD signaling, or induced chronic membrane depolarization. Chronic depolarization doubled the number of cells expressing neuronal markers produced with glutamatergic as well as with dopaminergic transcription factor cocktails. Inhibition of GSK-3beta; and SMAD signaling similarly doubled the yield of glutamatergic NiNs, and enabled induction of neuronal markers and morphological transformation in human fibroblasts.</p><p>In addition to soluble microenvironmental factors, the physical microenvironment can also strongly influence various cellular phenotypes. This list includes many phenotypes related to endocytosis - the transit mechanism of nanoparticulate gene carriers entering cells during non-viral transfection. As such, we set out to determine if patterned physical substrate topography could be used to increase non-viral transfection. We employed a high-throughput screen of micropitted substrate topographies, and indeed found that larger, denser micropits could support significantly higher transfection efficiencies in fibroblasts, compared to smooth substrates. The same topographies produced higher reverse transfection efficiencies, and greater siRNA-mediated knockdown of a reporter gene. The control of transfection with substrate topography is a new design parameter that could find broad application in in vitro non-viral reprogramming strategies, as well as in the intelligent design of nucleic acid-eluting scaffolds in vivo. </p><p>Encouraged by our success with direct neuronal reprogramming, and armed with a greater understanding of some microenvironmental mediators thereof, we attempted to produce non-virally-induced oligodendroglial progenitor cells (NiOPCs), which has been historically challenging for other investigators. We discovered the fibroblastic intracellular microenvironment is a significant barrier to the function of Olig2 - a master regulator of OPC phenotype - in direct reprogramming. Fibroblasts do not express Olig2 chaperones which are normally expressed in OPCs, causing Olig2 to become sequestered in the cytoplasm of transfected PMEFs. We relieved this barrier through fusion of a strong nuclear localization sequence (NLS) to Olig2, which repartitioned Olig2-NLS from the cytoplasm to the nucleus in transfected fibroblasts. The use of Olig2-NLS in iOPC reprogramming cocktails resulted in a drastic improvement in the yield of OPC-specific marker expression. The improvement associated with Olig2-NLS was insufficient to elicit significant myelin protein expression with the non-viral system, but the yield of virally-induced oligodendrocyte-like cells (iOLs) was improved dramatically. Further enhancements will be required to generate fully-reprogrammed NiOPCs, but the increased efficiency of viral iOPC generation is immediately useful for disease modeling and potentially in cell replacement therapies if human cells can be converted for the first time using this technique. During direct reprogramming, CNS-specific transcription factors are delivered to foreign intracellular contexts as a rule, which may reduce their ability to function effectively; we have shown this can be a critical yet under-appreciated determinant of the success or failure of a direct reprogramming system.</p><p>Taken together, the technological and intellectual advancements we describe herein represent significant improvements to non-viral transfection and reprogramming systems. These techniques can find broad appeal to the many researchers and clinicians deploying these systems. More specifically, we present significant steps towards realization of the dream of safe and effective autologous cell therapies against devastating and currently-intractable neurodegenerative diseases.</p><br>Dissertation

Fibroblasts are a heterogeneous cell population as testified by the absence of specific markers and by their documented retention of positional memory. Fibroblasts have recently gained much attention as they are the adult somatic cell most commonly used for reprogramming to induced pluripotent stem cells (iPSCs). iPSCs are somatic cells genetically reprogrammed to a pluripotent state that makes them resemble embryonic stem cells (ESCs). iPSCs are a very promising tool for regenerative medicine because of their autologous origin and their ability to differentiate towards cell of all three germ layers. However, the iPSCs reprogramming technology is still affected by variable and low efficiency. We hypothesize that the heterogeneity of fibroblasts may play a role in determining cell reprogramming. To test our hypothesis, we isolated and cultured human dermal fibroblasts (HDFs) from five different anatomic sites (abdomen, breast, neck, thigh and arm) and performed a comparative study analyzing their morphology, proliferation, apoptosis and ability to migrate. Further, we evaluated whether the expression of mesenchymal and epithelial markers, the ability to differentiate and release growth factors in culture medium varied among fibroblasts from different anatomic sites. Our results revealed that, although fibroblasts from different anatomic sites had similar morphology, all other features varied according to the region from which they were isolated. Furthermore, we observed similarity between fibroblasts and mesenchymal stem cells (MSCs) that also varied among HDFs populations. A low proliferation index, the release of specific growth factors, the expression of mesenchymal markers and the promptness to differentiate towards mesodermal lineages, might improve the efficiency of reprogramming. On these bases, abdomen dermal fibroblasts might represent the ideal candidates for reprogramming; since they have a low proliferation index, express high amount of mesenchymal markers, differentiate more precociously and release growth factors that improve the efficiency of reprogramming. Further the high expression of mesenchymal markers and their ability to differentiate more precociously suggest that fibroblasts isolated from abdomen skin could represent a less differentiated phenotype, more similar to MSCs than the other population of fibroblasts and probably easier to reprogram. Therefore, our data offer novel perspectives in the field of cell reprogramming, suggesting that the choice of the dermal population of fibroblasts might influence the efficiency of reprogramming itself.

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina<br>Estratégias de reprogramação celular têm evidenciado a flexibilidade do destino celulare, através de fatores de transcrição (FTs) específicos dos tipos celulares para converter células somáticas em pluripotentes. A reprogramação direta de um tipo de célula diferenciada em outr,o também foi demonstrada e explorada para elucidar os mecanismos de biologia celular e para fins de medicina regenerativa. Recentemente, demonstrámos que células dendríticas (CDs) apresentadoras de antigénios, podem ser reprogramadas em tipos de células diferentes,através da combinação de FTs. Classicamente, pensa-se que um progenitor específico da linhagem mielóide origina subgrupos de CDs funcionalmente diferentes: CDs convencionais (cCDs) são células apresentadoras de antigénios que desencadeiam respostas imunitárias; CDs plasmocitóides (pDCs) são células que produzem Interferão tipo I durante uma infecção viral. No entanto, os mecanismos exactos que regulam a divergência dos diferentes subtipos celulares durante o desenvolvimento das CDs ainda necessitam de ser estabelecidos. Recentemente identificámos os FTs Irf8, Pu.1 e Batf3 como suficientes e necessários para reprogramar fibroblastos em cCDs do tipo 1. Dado o papel importante dos FTs nas decisões de destino celular das diferentes CDs, o objetivo deste estudo é investigar a heterogeneidade das DCs através da caracterização mínima dos FTs e necessária para induzir DCs a partir de fibroblastos. Combinando a revisão bibliográfica e análise computacional, identificámos 23 TFs candidatos com potencial para originar pCDs, que demonstraram ter um papel importante na especificação de pCDs. Validámos em ratinho o repórter Clec9a que, é expresso em pCDs, através da proteína fluorescente tdTomato tornando este modelo adequado para o rastreamento de pCDs originadas pela sobreexpressão de FTs. Em seguida, fibroblastos embrionários de ratinho (MEFs) com o repórter Clec9a, foram transduzidos com um conjunto de FTs indutores de pCDs, usando um sistema lentiviral induzido por doxiciclina. Através da eliminação individual de cada TF, identificámos Irf8 e TF2 como a combinação mínima necessária para a activação do repórter. A expressão de moléculas de complexo de histocompatibilidade principal (MHC) de classe II, importante para a funcionalidade das DCs, também foi observada como dependente do Irf8 e TF2. Além disso, este estudo destacou o papel do TF1 na especificação das pCDs. Embora não seja intrinsecamente necessário, quando combinado com Irf8 e TF2, o TF1 aumenta a expressão de marcadores de superfície típicos de pCDs, que origina células tdTomato + B220 + Bst2 + pCD-like por reprogramação direta. Em resumo, este estudo fornece evidências de que o Irf8 quando combinado com o TF2 e o TF1 inicia um programa de reprogramação de fibroblastos em pCDs. Estas descobertas fornecem informações valiosas sobre a especificação de pCDs. No futuro, a geração de pCDs por reprogramação direta abre caminhos para a indução de respostas imunes antivirais através de células de engenharia autóloga.<br>Cellular reprogramming strategies have highlighted the flexibility of cell fates with the possibility to use cell-type-specific transcription factors (TFs) to convert somatic cells into pluripotency. Direct lineage conversions of one differentiated cell-type into another have also been demonstrated and explored for elucidating cell biology mechanisms and for regenerative medicine purposes. Recently, we have demonstrated that antigen presenting Dendritic cells (DCs) can be reprogrammed into unrelated cell-types by a small combination of TFs. Classically, it is thought that a myeloid DC committed progenitor gives rise to the functionally different DC subsets: conventional DCs (cDCs) are professional Antigen Presenting Cells (APC) driving antigen-specific immune responses; plasmacytoid DCs (pDCs) are professional producers of type I interferons during viral infection. However, the timing and exact mechanisms regulating the divergence of the different subsets during DC development is still to be established.We have recently identified Irf8, Pu.1 and Batf3 as sufficient and necessary to induce a cDC type 1 fate in fibroblasts. Given the important role of TFs in cell-fate decisions of the different DC subsets, the aim of this study is to investigate DC heterogeneity by fine-tuning the minimal TF network necessary to induce DC fate in order to program pDCs from fibroblasts.By combining literature review and computational analysis, we have identified 23 pDC-inducing candidate TFs with important roles in pDC specification and restricted pDC expression. We have then validated that in Clec9a reporter mouse, pDCs are labelled with tdTomato fluorescent protein making this model suitable for screening pDC-inducing factor. Then we have transduced Clec9a reporter mouse embryonic fibroblasts (MEFs) with a set of the pDC-inducing TFs using a doxycycline-induced lentiviral system. By sequential individual elimination of each TF, we have identified Irf8 and TF2 as the minimal combination required for reporter activation. Major histocompatibility complex (MHC) class II molecules’ expression important for DC functionality was also observed to be dependent of Irf8 and TF2. Moreover, our study highlighted the role of TF1 for pDC specification. Whilst not being intrinsically required, when combined with Irf8 and TF2, TF1 increases the expression of pDC- typical surface markers with the generation of tdTomato+ B220+ Bst2+ pDC-like cells by direct reprogramming. In summary, we provide evidence that Irf8 when combined with TF2 and TF1 kicks-start a pDC program in fibroblasts. These findings provide valuable insights into pDC specification. In the future, the generation of pDCs by direct reprogramming opens avenues for inducing anti-viral immune responses with autologous-engineered cells.

碩士<br>國立高雄大學<br>生物科技研究所<br>99<br>The somatic cell could reprogram to induced pluripotent stem cell (iPSC) by gene transfection vector. The characters of iPS cells are like embryonic stem cells (ESC), they have the same gene expression and life characteristic, include self-renew and differentiate to three germ layers. The preparation of iPSC is by lentiviral vector, but the transfect rate of is only 0.1 to 1%. Ultrasound is a new method of gene transfection. When ultrasound transmits energy in fluid, it could cause cavitations and damage the cell membrane. Therefore, ultrasound could enhance the cell permeability and then increase the gene transfecrion rate. In this study, different sizes of liposomes were used to mimic cells and lentivirus. The permeability of liposomes was increased during insonation. Same phenomena were found for applying sound waves on mouse fibroblast in presence of calcein-liposomes and GFP-lentiviral vectors. Sound waves could enhance the binding of calcein-liposomes and increase the transfection of GFP-lentiviral vectors to fibroblast. The transfection efficiency of GFP-lentivires is 23.81% which 2.27-folds higher than that without ultrasound. Same protocol was employed for the transfection of iPSC. The iPS cellular colonies number is 40% higher than without sound treatment. The expression of iPS cells is then identified by cellular morphology, DNA electrophoresis, immunofluroescence and Raman microscopy. The result showed that ultrasound could enhance lentiviral vector gene transfection and then increase iPS cells colonies.

碩士<br>國立成功大學<br>生物科技研究所碩博士班<br>94<br>Nuclear reprogramming is defined as altering the gene activity of differentiated cells to express the characteristics and function of different lineage, even resume their pluripotency. Several researches have demonstrated that nuclear reprogramming is possible. Nuclear transfer with somatic cells had proven to be able to give rise to cloning animals and derivation of embryonic stem cells. Induction of plasticity of adult stem cells could promote somatic cells to trans-differentiate into other cell lineages. 　After increasing permeability of the plasma membrane and introduction of ES-D3 cell extract, the morphology of somatic cells changed into a ES cell-like shape. Quantitative real-time PCR analysis showed both reprogrammed Dunni cells and HS-68 cells expressed Oct-4, Nanog and Pecam-1 genes which are stage-specific to undifferentiated ES cells. Strong alkaline phosphatase (AP) activity was also observed in reprogrammed cells. Under spontaneous differentiation, reprogrammed somatic cells could trans-differentiate into different lineages of three primary germ layers. According to our results, terminal differentiated human and mouse fibroblasts can be reprogrammed by mouse embryonic stem cell extracts. However, the protocol should be further modified to achieve a better efficiency on reprogramming. 　In the present study, we found that the cellular characteristics of somatic fibroblast cells derived from mouse skin (Dunni cells) and human foreskin (HS-68) could be reprogrammed by the cell extract of mouse embryonic stem cells (ES-D3).

碩士<br>國立臺灣海洋大學<br>生物科技研究所<br>98<br>Epithelial cell adhesion molecule (EpCAM) is a cell adhesion molecule and a potential target for therapeutic antibodies. Recently, EpCAM has been shown to regulate tumor cell proliferation by its nucleocytoplasmic intracellular domain fragment (ICD). EpCAM intracellular domain (EpICD) activates C terminal myelocytomatosis oncogene (c-Myc) in nucleus by Wnt signal pathway. On the other hand, the complex of EpCAM and the tight junction protein claudin-7 (CLDN-7) would promote tumorigenicity and accelerates tumor growth. We observed that EpCAM and CLDN-7 express in both mouse embryonic stem cells and induced poluripotent stem cells. In this study we first discovered that the expressions of EpCAM and CLDN-7 would be activated when reprogramming mouse embryonic fibroblast cells into induced pluripotent stem cells. The efficiency of iPS reprogramming was enhanced by overexpressing EpCAM, CLDN-7 or both. Furthermore, silence of endogenous EpCAM or CLDN-7 resulted in the reduction of iPS reprogramming. However, ectopic expression of EpCAM and CLDN-7 in differential somatic cells such as mouse fibroblast cells had no effect on the activation of pluripotency-associated gene Nanog, Oct4, Sox2, c-Myc and KLF4. Our data show that EpCAM positively regulates iPS reprogramming, yet the detail mechanism remains unclear.

Tese de mestrado, Engenharia Biomédica e Biofísica (Engenharia Clínica e Instrumentação Médica) Universidade de Lisboa, Faculdade de Ciências, 2017<br>Foi recentemente desenvolvido um novo método revolucionário capaz de reprogramar fibroblastos em células pluripotentes induzidas através da expressão de 4 fatores de transcrição (Oct4, Sox2, Klf4 e c-Myc). A reprogramação de fibroblastos em células pluripotentes induzidas (iPSC) foi um grande avanço científico com possíveis aplicações clínicas e fins terapêuticos, no entanto, à medida que as células diferenciadas (células somáticas) vão envelhecendo devido à acumulação de marcas genéticas e epigenéticas, estas tornam-se mais resistentes à sua conversão para um estado pluripotente. Posto isto, um dos principais objetivos deste projeto passava por perceber o impacto que o envelhecimento tem na reprogramação de células humanas em células estaminais pluripotentes induzidas humanas (hiPSCs). De facto, ao reprogramar fibroblastos adultos de ratinho em células pluripotentes induzidas (miPSCs – mouse induced pluripotent stem cells), observou-se que o envelhecimento celular estava a atuar como uma barreira, reduzindo a eficiência da reprogramação celular. Curiosamente, nenhuma correlação entre a eficiência da reprogramação celular e o envelhecimento foi observada na reprogramação de células humanas. Ao realizar a reprogramação celular de fibroblastos embrionários humanos (WI38) e fibroblastos humanos com 3 anos (3yr) com baixa passagem (passage 4), observou-se o mesmo número de células hiPSCs geradas. Sugerindo assim, e ao contrário do esperado e observado em células de ratinho, que a idade das células humanas utilizadas para formar hiPSCs não dificulta de forma significativa a reprogramação celular. Contudo, foi observado que o número de passagens das células em cultura tinha uma contribuição importante na eficiência da reprogramação celular de fibroblastos humanos em hiPSCs. Ao tentar reprogramar fibroblastos humanos com uma baixa passagem (passagem 4) e uma alta passagem (passagem 7), e apesar de as células com passagem 7 expressarem níveis mais elevados de hOCt4, apenas as células humanas com uma baixa passagem reprogramaram. Estes resultados sugerem que o número de passagens celulares tem uma contribuição importante na eficiência da reprogramação celular. De facto, estes resultados podem ser explicados pela simples razão de que cada passagem celular realizada em cultura, aumenta o risco de ocorrer dano no ADN, mutações e ainda alterações nas características celulares originando assim, alterações na morfologia, na resposta a estímulos, na taxa de crescimento, na expressão de proteínas e na eficiência da transfecção celular. De acordo com Leonard Hayflick e Paul Moorhead, as células humanas têm um número limitado de divisões celulares em cultura que poderá variar entre tipos de células. Cada divisão celular pode induzir um encurtamento dos telómeros que poderá resultar em senescência. Podendo esta ser ainda induzida através de dano molecular que ocorre de forma aleatória, pelo stress oxidativo e pela danificação do ADN. A senescência pode ainda ser acumulada em alguns tecidos contribuindo para a disfunção orgânica. Sugere-se portanto, que devido ao número elevado de passagens celulares, os fibroblastos embrionários humanos e os fibroblastos humanos com 3 anos sofreram o encurtamento dos seus telomeros, levando assim, a um fenótipo senescente, reduzindo a eficiência do processo de reprogramação celular em células humanas. Um outro objetivo deste projeto passava por encontrar outras estratégias celulares que ajudem a ultrapassar a limitação do envelhecimento na reprogramação celular de células humanas, através da modulação de RNAs longos não codificantes (lncRNA). No entanto, como os resultados referentes ao envelhecimento demonstraram uma não influência na eficiência da reprogramação de células humanas em hiPSCs, decidiu-se desvendar e entender, qual a função do lncRNA Zeb2NAT na reprogramação celular e qualidade de células humanas. Embora, já reportado anteriormente pelo nosso laboratório, que a supressão do lncRNA Zeb2NAT em células de ratinho, utilizando olignucleótidos contra-senso (anti sense), denominados por LNAs aumentam significativamente a reprogramação celular de fibroblastos de ratinho envelhecidos ajudando assim contornar as barreiras mesenquimais, ainda nada se sabia sobre o impacto que a diminuição do Zeb2 e do Zeb2NAT poderia ter na reprogramação celular de células humanas. Contudo, utilizando a mesma abordagem, acima descrita, em fibroblastos embrionários humanos e em fibroblastos humanos com 3 anos, verificou-se um atraso na formação de hiPSCs das células humanas que sofreram uma desregulação tanto do Zeb2 como do Zeb2NAT, comparativamente às células do controlo e do LNA-controlo (um LNA não específico a nenhuma sequência genómica humana, utilizado como um controlo negativo da transfeção de LNAs). De facto, 14 dias após a primeira transfeção com LNAs em ambas as duas linhas celulares, apenas começaram a formar-se hiPSCs no controlo e no LNA-controlo, tendo o controlo um número maior de hiPSCs formadas em ambas as células, comparativamente à condição com LNA-controlo. No entanto, 19 dias e 35 dias depois da primeira transfeção, as WI38 com a desregulação do Zeb2 e do Zeb2NAT, respetivamente, começaram a reprogramar. Nenhuma formação de hiPSCs nas células humanas de 3 anos com a desregulação do Zeb2 e do Zeb2NAT foi observada. Tendo em consideração que as células de ratinho após diminuição dos níveis do Zeb2 e do Zeb2-NAT começaram a reprogramar de forma mais eficiente que o controlo e o LNA-controlo, estes resultados em células humanas sugerem que a desregulação do Zeb2 e do Zeb2NAT pode estar, de certa maneira, a atrasar e até mesmo a atuar como um bloqueador da reprogramação celular humana. Este bloqueio/atraso que se observou na reprogramação de fibroblastos humanos em hiPSCs poderia ter como principal responsável a transfeção de LNAs, necessária para diminuir a expressão do Zeb2 e do Zeb2NAT. De facto, já foi demonstrado que o uso do reagente de transfeção Lipofectamine ativa algum stress nos genes afetando o ciclo da regulação e/ou a sinalização metabólica nas células. No entanto, as células com a condição do LNA-controlo reprogramaram com a mesma rapidez que o controlo (sem LNAs). E a diferença do número de hiPSCs geradas pelo LNA-controlo, comparativamente ao controlo, acaba por não ser significativamente diferente. Isto sugere, que embora a transfeção de LNAs tenha um impacto na reprogramação celular, diminuindo a eficiência desta, existe um outro fator que está a contribuir para que as células com a diminuição da expressão do Zeb2 e do Zeb2-NAT tenham uma redução na eficiência da reprogramação celular em células humanas. Todavia, ainda é incerto a razão de a desregulação do Zeb2 e do Zeb2NAT terem atrasado e até mesmo bloqueado a reprogramação celular em células humanas. É possível que possa ser devido ao mecanismo que rege o Zeb2 que, de alguma forma, é diferente comparativamente ao mecanismo observado em ratinhos. Outra possibilidade a ter em conta é a necessidade de realizar uma otimização ao protocolo da transfeção de LNAs, de forma a adaptar esta ao protocolo da reprogramação celular. Apesar disto, acreditamos que esta abordagem constitui uma nova estratégia para estudar o impacto dos lncRNAs na reprogramação celular e antecipamos ainda que os resultados produzidos irão gerar contribuições importantes na área de investigação do envelhecimento e da reprogramação celular.<br>Revolutionary progress has been achieved recently following the discovery of cellular reprogramming by the expression of a combination of 4 transcription factors (Oct4, Sox2, Klf4 and c-Myc). The reprogramming of fibroblasts to induced pluripotent stem cells (iPSC) was a major scientific advance however, as differentiated cells grow old and due to the accumulation of genetic and epigenetic marks, they become more resistant to be converted back to a pluripotent state. In fact, when tried to reprogram mice cells into miPSCs, aging was acting as a barrier, reducing the efficiency of reprogramming aged cells. However, through our recent findings no correlation between cellular reprogramming efficiency and aging was found in human cells. However, we observed that the number of passages had an important contribution in the efficiency of reprogramming human fibroblasts into hiPSCs. Reprogramming experiments with human fibroblasts with lower passage (passage 4) and high passage (passage 7) show that only the human cells with a lower passage, reprogrammed. The higher the number of passages in vitro, the lower the efficiency of cellular reprogramming of human cells. The other main interest of this project converged on cellular strategies to overcome this aging limitation by modulating long noncoding RNAs (lncRNAs). However, as the results showed a non-influence of aging on reprogramming human cells into hiPSCs it was decided to focus on understanding exactly the role of the lncRNA Zeb2NAT on cellular reprogramming of human cells. As previously reported by our lab, the suppression of Zeb2NAT in mice cells significantly increased the reprogramming of aged fibroblasts. However, when tested the same approach in human cells, a delay in generating hiPSCs was observed, suggesting that the knockdown of Zeb2 and Zeb2NAT can be acting as a blocker of cellular reprogramming in human cells, going against what was observed in mice cells. Still, it’s uncertain why downregulation of Zeb2 and Zeb2NAT were delaying and blocking the cellular reprogramming in human cells. It is possible that the mechanisms of Zeb2 in humans are, somehow, different from those observed in mice. Another possibility to take in account can be due the fact the protocol isn´t fully optimized. Despite this, we believe this approach constitutes a novel strategy to study the impact of lncRNAs in cellular reprogramming and we anticipate that the output of this proposal will generate important contributions to the aging and cellular reprogramming research field.

Indiana University-Purdue University Indianapolis (IUPUI)<br>Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.