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Différenciation des cellules souches embryonnaires humaines vers l'hépatocyte / Production of hepatocytes from human embryonic stem cellsFunakoshi, Natalie 06 December 2011 (has links)
Les hépatocytes humains adultes en culture primaire (HHCP) ont de nombreuses applications en physiopathologie hépatique, en pharmacologie et en biothérapie, mais sont limitées par leur faible disponibilité. Les cellules souches embryonnaires humaines (hES) sont une source prometteuse pour l'obtention d'hépatocytes en grande quantité. Nous avons développé un modèle in vitro de différenciation de hES en hépatocytes en reproduisant toutes les étapes de l'ontogenèse hépatique. Au cours de la différenciation, l'expression de 41 gènes marqueurs du foie a été étudiée et comparée aux HHCP, au foie fœtal et aux progéniteurs hépatiques issus du foie adulte. Les résultats démontrent qu'au bout de 21 jours de différenciation, les cellules souches embryonnaires différenciées en hépatocytes (hES-Hep) ont atteint un état de maturation équivalente aux hépatocytes fœtaux aux alentours de 20 semaines de gestation. L'expression forcée du xénorécepteur CAR dans les hES-Hep a induit l'expression des gènes de la détoxification et la biotransformation de midazolam, un substrat de CYP3A4. Ces résultats pourront contribuer au développement de cultures de hES-Hep comme alternative aux HHCP pour les études du métabolisme des xénobiotiques et pour la thérapie cellulaire. / Primary cultures of human adult hepatocytes (PCHH) have widespread potential applications in liver physiopathology , pharmacology, and cell-based therapies, but are currently limited by poor availability. Human embryonic stem cells (hES) are a promising source for the generation of hepatocytes in large quantities. In this study, we differentiated hES into hepatocytes by mimicking in vitro the various stages of hepatic ontogenesis. We analyzed the expression of a panel of 41 liver marker genes in hepatocyte-like cells derived from hES (hES-Hep) in comparison with PCHH, fetal liver and progenitors obtained from adult liver. The data revealed that after 21 days of differentiation ES-Hep are representative of fetal hepatocytes at around 20 weeks of gestation. The forced expression of the xenoreceptor CAR in hES-Hep induced the expression of detoxification genes as well as the biotransformation of midazolam, a substrate of CYP3A4. These results may contribute to the development of hES-Hep cultures as an alternative to PCHH for studies of xenobiotic metabolism and for cell-based therapies.
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L’enzyme CD10 : un acteur clé dans l’identification et la régulation des cellules souches mammaires humaines / The CD10 enzyme is a key player to identify and regulate human mammary stem cellsCascales, Élodie 17 December 2010 (has links)
Dans différents types de cancers, notamment dans le cancer du sein, il existe une population cellulaire à l’origine des mécanismes de rechute de la maladie plusieurs années après la fin des traitements initiaux, caractérisée par des propriétés de cellules souches et une chimiorésistance unique dont le mécanisme est inconnu. Pour ces raisons il est important de comprendre les mécanismes et de connaître les acteurs physiologiques impliqués à la fois dans la régulation des cellules souches normales et cancéreuses. Le CD10 est une endopeptidase zinc-dépendante capable d’inactiver un certain nombre de peptides impliqués, entre autre, dans le développement de la glande mammaire. Nos recherches ont permis de montrer que la population cellulaire exprimant le CD10 dans la glande mammaire était enrichie en cellules souches/progéniteurs communs précoces/cellules myoépithéliales. Nos résultats suggèrent que l’adhérence des cellules souches au stroma via l’intégrine β1 et le clivage de certains peptides par le CD10 sont des éléments clés du micro-environnement permettant le maintien du réservoir de cellules souches et de progéniteurs précoces dans la glande mammaire. Le tissu adipeux est également un des constituants majeur de l’environnement de la glande mammaire et joue un rôle dans la sécrétion de facteurs de croissances également impliqués dans l’homéostasie du tissu mammaire. Nos résultats ont suggéré qu’en plus de son rôle nourricier, le tissu adipeux pouvait constituer une source potentielle de cellules souches épithéliales luminales pouvant ainsi être considérée comme une nouvelle source cellulaire à l’origine de certains cancers du sein. / In breast, the existence of cancer stem cells has been demonstrated and that explain a number of observations as tumour heterogeneity. Other studies have demonstrated the resistance of radio and chemotherapy by different innate or acquired stem cell specific mechanisms that could explain relapse few years after the traitment. For all these reasons, that is very important to understand these mechanisms and to know physiological actors both implicated in the regulation of normal or cancer stem cells. CD10 is a zinc-dependant metallo-endopeptidase that inactivates a number of signalling peptides that could be implicated in mammary growth and differentiation. We have showed that CD10+ cell sorted population is enriched in Stem Cells/Early Common Progenitors/MyoEPithelial cells. We demonstrate that the protease activity of CD10 and the adhesion function of beta1-integrin are required to prevent differentiation of mammary stem cells/early progenitors. Taken together, our data suggest that integrin-mediated contact with the basement membrane and cleavage of signalling factors by CD10 are key elements in the microenvironment that maintains the progenitor and stem cell pools in the mammary gland. Adipose tissue is also a major component of the mammary microenvironment implicated in its homeostasis by the secretion of soluble factors. Our results suggested that the adipose tissue could be considered as a potential source of stem cells that differentiated into the luminal epithelial lineage involved in some breast cancers.
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Optogenetic Differentiation of Cardiovascular Cells from Pluripotent Stem CellsPeter Benjamin Hellwarth (10223837) 29 April 2021 (has links)
<p>Stem cell technologies hold great
promise in solving problems within fields such as drug development,
regenerative medicine, and disease modeling. Stem cell engineering provides a
mechanism that will help stem cells achieve this promise. Currently, many applications
within tissue engineering are limited by a lack of ability to create accurate
micro-physiological structures that recapitulate multicellular tissue patterns <i>in
vivo</i>. Precise control of spatial and temporal signaling is desired to
perform concurrent differentiation to multiple cell types intentionally. The
OptoWnt construct, a novel optogenetic system activating the Wnt signaling pathway,
achieves precise spatiotemporal regulation, in pursuit of greater control in stem
cell differentiation. We utilize OptoWnt, to differentiate stem cells into cardiovascular
cells: endothelial progenitor cells and cardiomyocytes, valuable cell types for
designing microtissues. Endothelial
cells comprise the luminal lining of blood and lymphatic vessels, providing the
integral structure for distribution within the body, separating mobile and
stationary tissues. Cardiomyocytes provide the force required to pump blood
throughout the human body and are a highly desired cell type in regenerative
medicine.</p>
<p>In this project, we have applied
an optogenetic induced signaling pathway, OptoWnt, to differentiate human pluripotent
stem cells (hPSCs) into cardiovascular cells via light-induced activation of
Wnt signaling pathway. In the analysis of these cells and comparison to
previous small molecule approaches to cardiovascular cell differentiation, we
demonstrate the robustness of the optogenetic approach and similar efficiency
that it has with the small molecule approach. In short, we have further
demonstrated the utility and potential of optogenetic induction of
developmental pathways, via the OptoWnt construct.</p>
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Desenvolvimento de metodologia radiográfica e volumétrica dos diferentes estágios de desenvolvimento dentário para qualificação de material biológico em Engenharia Tecidual / Development of radiographic and volumetric metodologies from diferentes tooth development stages as a qualification for harvesting biological material for Tissue EngeneeringDuailibi Neto, Eduardo Felippe 12 March 2013 (has links)
A utilização de Células-tronco e técnicas da Engenharia Tecidual representa um grande avanço tecnológico e beneficiará muitos pacientes com suas conquistas. A descoberta de germes dentários como uma fonte confiável de células-tronco possibilitou diversas pesquisas nesta área. Duailibi et al. (2011) sugeriram uma nova classificação de desenvolvimento dentário baseada pela quantidade de material biológico coletado indicando a necessidade de métodos de diagnóstico por imagem para esta nova classificação. Na literatura diversos trabalhos indicam métodos de classificação dentária e métodos para estimar a idade fisiológica de indivíduos. O presente estudo tem o objetivo de adaptar alguns destes métodos para estimar o estágio de desenvolvimento proposto por Duailibi et al. (2011) consequentemente indicando a quantidade de células-tronco esperadas nas amostras. Para tanto, submeteu-se uma coleção de 67 dentes previamente classificados por Duailibi et al. (2011) à técnica rpcl e à tcfc para a obtenção de imagens e a aplicação de técnicas de estimativas por proporções lineares e volumétricas. Os resultados por análises lineares indicaram valores de R2 para o método de proporção de comprimento CDCP de 0,14050; CCCP de 0,65369; CCCR de 0,5408; CDCR de 0,54074; o método de proporção de área APAD de 0,23925; e método de proporção de volume VPVD de 0,08553, com valor de p menor ou igual à 0,05. Concluindo este estudo indica-se o método de rpcl utilizando a análise do comprimento entre coroa e polpa como o mais indicado para estimar o estágio de desenvolvimento. / The usage of human dental stem cells and tissue engineering technics represents a huge tecnological development and it may benefits many patients in a promissing future. The discovery of suitable source of human dental stem cells were made using tooth buds. Duailibi et al. 2011 indicated a new tooth classification on a stem cell harvesting based research, sugesting new methods for diagnosis these stages. Several method were developed for dental age assesement. The presente study aims to evaluate some of these dental age technics and make adaptations for estimating Duailibi et al. 2011 tooth stages. A 67 tooth sample previoulsy classificated by Duailbi et al. 2011 were submited through periapical parallel long cone X-rays and CBCT analysis. Age estimation ratio methods were applied by measuring tooth/root lenth, crown/root lenth, tooth/pulp lenth, crown/pulp lenth, tooth/poulp área and tooth/pulp volume. Results indicated by linear regression analisys a R2 value of tooth/pulp lenth 0,14050; crown/pulp lenth 0,65369; crown/root lenth 0,5408; tooth/root lenth 0,54074; pulp/tooth volume 0,23925; e tooth/pulp volume de 0,08553, with p value of 0,005. In conclusion , the best method for estimating Duailibi et al. 2011 tooth classification techinic is made by using periapical long cone X-rays using crown/pulp lenth ratio.
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Desenvolvimento de metodologia radiográfica e volumétrica dos diferentes estágios de desenvolvimento dentário para qualificação de material biológico em Engenharia Tecidual / Development of radiographic and volumetric metodologies from diferentes tooth development stages as a qualification for harvesting biological material for Tissue EngeneeringEduardo Felippe Duailibi Neto 12 March 2013 (has links)
A utilização de Células-tronco e técnicas da Engenharia Tecidual representa um grande avanço tecnológico e beneficiará muitos pacientes com suas conquistas. A descoberta de germes dentários como uma fonte confiável de células-tronco possibilitou diversas pesquisas nesta área. Duailibi et al. (2011) sugeriram uma nova classificação de desenvolvimento dentário baseada pela quantidade de material biológico coletado indicando a necessidade de métodos de diagnóstico por imagem para esta nova classificação. Na literatura diversos trabalhos indicam métodos de classificação dentária e métodos para estimar a idade fisiológica de indivíduos. O presente estudo tem o objetivo de adaptar alguns destes métodos para estimar o estágio de desenvolvimento proposto por Duailibi et al. (2011) consequentemente indicando a quantidade de células-tronco esperadas nas amostras. Para tanto, submeteu-se uma coleção de 67 dentes previamente classificados por Duailibi et al. (2011) à técnica rpcl e à tcfc para a obtenção de imagens e a aplicação de técnicas de estimativas por proporções lineares e volumétricas. Os resultados por análises lineares indicaram valores de R2 para o método de proporção de comprimento CDCP de 0,14050; CCCP de 0,65369; CCCR de 0,5408; CDCR de 0,54074; o método de proporção de área APAD de 0,23925; e método de proporção de volume VPVD de 0,08553, com valor de p menor ou igual à 0,05. Concluindo este estudo indica-se o método de rpcl utilizando a análise do comprimento entre coroa e polpa como o mais indicado para estimar o estágio de desenvolvimento. / The usage of human dental stem cells and tissue engineering technics represents a huge tecnological development and it may benefits many patients in a promissing future. The discovery of suitable source of human dental stem cells were made using tooth buds. Duailibi et al. 2011 indicated a new tooth classification on a stem cell harvesting based research, sugesting new methods for diagnosis these stages. Several method were developed for dental age assesement. The presente study aims to evaluate some of these dental age technics and make adaptations for estimating Duailibi et al. 2011 tooth stages. A 67 tooth sample previoulsy classificated by Duailbi et al. 2011 were submited through periapical parallel long cone X-rays and CBCT analysis. Age estimation ratio methods were applied by measuring tooth/root lenth, crown/root lenth, tooth/pulp lenth, crown/pulp lenth, tooth/poulp área and tooth/pulp volume. Results indicated by linear regression analisys a R2 value of tooth/pulp lenth 0,14050; crown/pulp lenth 0,65369; crown/root lenth 0,5408; tooth/root lenth 0,54074; pulp/tooth volume 0,23925; e tooth/pulp volume de 0,08553, with p value of 0,005. In conclusion , the best method for estimating Duailibi et al. 2011 tooth classification techinic is made by using periapical long cone X-rays using crown/pulp lenth ratio.
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