Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix
Autor(a) principal: | |
---|---|
Data de Publicação: | 2018 |
Tipo de documento: | Tese |
Idioma: | eng |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da USP |
Texto Completo: | http://www.teses.usp.br/teses/disponiveis/10/10132/tde-31072018-161001/ |
Resumo: | Researches from different fields around the world are searching for both new sources of biomaterials and potential hepatocytes in order to supply drug tests, cell therapies, and cell transplantation as alternative therapeutic support to liver diseases and injuries. Placenta may be eligible as a new model in tissue engineering due to its rich extracellular matrix (ECM) and availability after birth. Placental scaffolds were produced by decellularization with 0.01, 0.1 and 1% SDS, and 1% Triton X-100 which were valued by means of structure and composition. Afterwards, placental scaffolds were co-cultured with mouse embryonic fibroblasts in a tridimensional (3D) rotating system. Placental scaffolds presented a well-preserved acellular ECM containing 9.42 ± 5.2 ng dsDNA per mg of ECM. Weak collagen I of the natives clearly appears in decellularized ECM while the collagen III, once well observed in native placenta, it was absent on scaffolds. This interesting observation may have been due to the solubilization SDS-induced of the collagen III fibrils during decellularization. Fibronectin was well-observed in placental scaffolds whereas laminin and collagen IV were strongly stained. Recellularized with fibroblasts by a 3D culture system, placental scaffolds showed potential for repopulation, with cells adhered throughout its acellular ECM. Placental scaffolds were then newly recellularized, aiming now for differentiation of mouse embryonic stem cells into hepatic cells. In a protocol of 23 days, it was simulated major events of liver embryonic development by adding growth factors. As result, a high index of cells adhered, proliferated and migrated throughout outer and inner scaffolds ECM surface. Absence of Oct4 and Nanog showed that Activin A and Wnt3a (d0-6) induced primitive endoderm fate, and negative label for Foxa2 and Sox17 representing BMP4 and FGF2 (d6-10) differentiation-induced generating definitive endoderm cells. Also, FGF1, FGF4 and FG8b (d10-14) induced hepatoblast phenotype cells, that were observed positive for AFP and CK7 markers. Finally, HGF and FS-288 (d14-23) induced to hepatocyte-like cells, positive for CK18 and Alb markers. The hepatocyte-like cells functional aspects were observed by glycogen storage. Though a heterogeneous cell hepatic lineage was confirmed, mouse placental scaffolds shown a useful model to support recellularization with simultaneous differentiation into hepatic fate simulating phases of embryonic development. |
id |
USP_2d89b1ea745a1d3267976734927bfdb4 |
---|---|
oai_identifier_str |
oai:teses.usp.br:tde-31072018-161001 |
network_acronym_str |
USP |
network_name_str |
Biblioteca Digital de Teses e Dissertações da USP |
repository_id_str |
2721 |
spelling |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrixAdesão e modulação da progênie hepatocitária de células-tronco embrionárias de camundongos sobre a matriz extracelular placentária de camundongosCélulas hepatocyte-likeCélulas-tronco embrionáriasCultura rotativa 3DEmbryonic stem cellsHepatocyte-like cellsMouse placental scaffoldsRecellularizationRecelularizaçãoRotating 3D cultureScaffolds placentários de camundongosResearches from different fields around the world are searching for both new sources of biomaterials and potential hepatocytes in order to supply drug tests, cell therapies, and cell transplantation as alternative therapeutic support to liver diseases and injuries. Placenta may be eligible as a new model in tissue engineering due to its rich extracellular matrix (ECM) and availability after birth. Placental scaffolds were produced by decellularization with 0.01, 0.1 and 1% SDS, and 1% Triton X-100 which were valued by means of structure and composition. Afterwards, placental scaffolds were co-cultured with mouse embryonic fibroblasts in a tridimensional (3D) rotating system. Placental scaffolds presented a well-preserved acellular ECM containing 9.42 ± 5.2 ng dsDNA per mg of ECM. Weak collagen I of the natives clearly appears in decellularized ECM while the collagen III, once well observed in native placenta, it was absent on scaffolds. This interesting observation may have been due to the solubilization SDS-induced of the collagen III fibrils during decellularization. Fibronectin was well-observed in placental scaffolds whereas laminin and collagen IV were strongly stained. Recellularized with fibroblasts by a 3D culture system, placental scaffolds showed potential for repopulation, with cells adhered throughout its acellular ECM. Placental scaffolds were then newly recellularized, aiming now for differentiation of mouse embryonic stem cells into hepatic cells. In a protocol of 23 days, it was simulated major events of liver embryonic development by adding growth factors. As result, a high index of cells adhered, proliferated and migrated throughout outer and inner scaffolds ECM surface. Absence of Oct4 and Nanog showed that Activin A and Wnt3a (d0-6) induced primitive endoderm fate, and negative label for Foxa2 and Sox17 representing BMP4 and FGF2 (d6-10) differentiation-induced generating definitive endoderm cells. Also, FGF1, FGF4 and FG8b (d10-14) induced hepatoblast phenotype cells, that were observed positive for AFP and CK7 markers. Finally, HGF and FS-288 (d14-23) induced to hepatocyte-like cells, positive for CK18 and Alb markers. The hepatocyte-like cells functional aspects were observed by glycogen storage. Though a heterogeneous cell hepatic lineage was confirmed, mouse placental scaffolds shown a useful model to support recellularization with simultaneous differentiation into hepatic fate simulating phases of embryonic development.Pesquisas de diferentes campos ao redor do Mundo estão em busca de novas fontes tanto de biomateriais, quanto de potenciais hepatócitos, a fim de suprir testes de drogas, terapias celulares e transplante de células, como suporte terapêutico alternativo para doenças e lesões hepáticas. Placentas podem ser elegíveis como um novo modelo em Engenharia Tecidual em decorrência de sua rica matriz extracelular (ECM), e disponibilidade após o nascimento. Os scaffolds placentários foram produzidos por decelularização com SDS 0,01, 0,1 e 1% e Triton X-100 1%, os quais foram avaliados por meio da estrutura e composição. Posteriormente, os scaffolds placentários foram co-cultivados com fibroblastos embrionários de camundongos em um sistema rotativo tridimensional (3D). Os scaffolds placentários apresentaram uma MEC acelular bem conservada, contendo 9,42 ± 5,2 ng/dsDNA/mg/MEC. O fraco colágeno I nos nativos aparece claramente na MEC descelularizada, enquanto o colágeno III bem visível na placenta nativa estava ausente nos scaffolds. Esta observação interessante pode decorrido da solubilização das fibrilas de colágeno III, induzida pelo SDS durante a decelularização. A fibronectina foi bem observada nos scaffolds placentários, enquanto a laminina e o colágeno IV estiveram fortemente marcados. Recelularizados com fibroblastos por um sistema de cultura 3D, os scaffolds placentários mostraram potencial para repovoamento, com células aderidas ao longo de sua MEC acelular. Os scaffolds placentários foram então novamente recelularizados, visando agora a diferenciação de células tronco-embrionárias de camundongos em células hepáticas. Em um protocolo de 23 dias, foram simulados os grandes eventos do desenvolvimento embrionário do fígado, pela adição de fatores de crescimento. Como resultado, um alto índice de células aderiu, proliferou e migrou através das superfícies externa e interna dos scaffolds. A ausência de Oct4 e Nanog demostraram que o Activin A e o Wnt3a (d0-6) induziram o destino endoderma primitivo, e a marcação negativa para Foxa2 e Sox17 representaram a geração de células endodermais definitivas pela diferenciação induzida por BMP4 e FGF2 (d6-10). Ainda, FGF1, FGF4 e FG8b (d10-14) induziram células do fenótipo hepatoblasto, que foram observadas positivas para os marcadores AFP e CK7. Finalmente, HGF e FS-288 (d14-23) induziram as células hepatocyte-like, positivas para os marcadores CK18 e Alb. The hepatocyte-like cells functional aspects were observed by glycogen storage. Though a heterogeneous cell hepatic lineage was confirmed, mouse placental scaffolds shown a useful model to support recellularization with simultaneous differentiation into hepatic fate simulating phases of embryonic development. Os aspectos funcionais das células hepatocyte-like foi observada pelo armazenamento de glicogênio. Embora uma linhagem hepática formada por células heterogêneas tenha sido confirmada, os scaffolds placentários de camundongos se mostraram um modelo útil para sustentar a recelularização com simultânea diferenciação em destino hepático, simulando fases do desenvolvimento embrionário.Biblioteca Digitais de Teses e Dissertações da USPMiglino, Maria AngélicaRomagnolli, Patricia2018-02-26info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttp://www.teses.usp.br/teses/disponiveis/10/10132/tde-31072018-161001/reponame:Biblioteca Digital de Teses e Dissertações da USPinstname:Universidade de São Paulo (USP)instacron:USPLiberar o conteúdo para acesso público.info:eu-repo/semantics/openAccesseng2020-08-22T16:00:06Zoai:teses.usp.br:tde-31072018-161001Biblioteca Digital de Teses e Dissertaçõeshttp://www.teses.usp.br/PUBhttp://www.teses.usp.br/cgi-bin/mtd2br.plvirginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.bropendoar:27212020-08-22T16:00:06Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)false |
dc.title.none.fl_str_mv |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix Adesão e modulação da progênie hepatocitária de células-tronco embrionárias de camundongos sobre a matriz extracelular placentária de camundongos |
title |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix |
spellingShingle |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix Romagnolli, Patricia Células hepatocyte-like Células-tronco embrionárias Cultura rotativa 3D Embryonic stem cells Hepatocyte-like cells Mouse placental scaffolds Recellularization Recelularização Rotating 3D culture Scaffolds placentários de camundongos |
title_short |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix |
title_full |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix |
title_fullStr |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix |
title_full_unstemmed |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix |
title_sort |
Adhesion and modulation of mouse embryonic stem cells hepatocyte progeny on mouse placental extracellular matrix |
author |
Romagnolli, Patricia |
author_facet |
Romagnolli, Patricia |
author_role |
author |
dc.contributor.none.fl_str_mv |
Miglino, Maria Angélica |
dc.contributor.author.fl_str_mv |
Romagnolli, Patricia |
dc.subject.por.fl_str_mv |
Células hepatocyte-like Células-tronco embrionárias Cultura rotativa 3D Embryonic stem cells Hepatocyte-like cells Mouse placental scaffolds Recellularization Recelularização Rotating 3D culture Scaffolds placentários de camundongos |
topic |
Células hepatocyte-like Células-tronco embrionárias Cultura rotativa 3D Embryonic stem cells Hepatocyte-like cells Mouse placental scaffolds Recellularization Recelularização Rotating 3D culture Scaffolds placentários de camundongos |
description |
Researches from different fields around the world are searching for both new sources of biomaterials and potential hepatocytes in order to supply drug tests, cell therapies, and cell transplantation as alternative therapeutic support to liver diseases and injuries. Placenta may be eligible as a new model in tissue engineering due to its rich extracellular matrix (ECM) and availability after birth. Placental scaffolds were produced by decellularization with 0.01, 0.1 and 1% SDS, and 1% Triton X-100 which were valued by means of structure and composition. Afterwards, placental scaffolds were co-cultured with mouse embryonic fibroblasts in a tridimensional (3D) rotating system. Placental scaffolds presented a well-preserved acellular ECM containing 9.42 ± 5.2 ng dsDNA per mg of ECM. Weak collagen I of the natives clearly appears in decellularized ECM while the collagen III, once well observed in native placenta, it was absent on scaffolds. This interesting observation may have been due to the solubilization SDS-induced of the collagen III fibrils during decellularization. Fibronectin was well-observed in placental scaffolds whereas laminin and collagen IV were strongly stained. Recellularized with fibroblasts by a 3D culture system, placental scaffolds showed potential for repopulation, with cells adhered throughout its acellular ECM. Placental scaffolds were then newly recellularized, aiming now for differentiation of mouse embryonic stem cells into hepatic cells. In a protocol of 23 days, it was simulated major events of liver embryonic development by adding growth factors. As result, a high index of cells adhered, proliferated and migrated throughout outer and inner scaffolds ECM surface. Absence of Oct4 and Nanog showed that Activin A and Wnt3a (d0-6) induced primitive endoderm fate, and negative label for Foxa2 and Sox17 representing BMP4 and FGF2 (d6-10) differentiation-induced generating definitive endoderm cells. Also, FGF1, FGF4 and FG8b (d10-14) induced hepatoblast phenotype cells, that were observed positive for AFP and CK7 markers. Finally, HGF and FS-288 (d14-23) induced to hepatocyte-like cells, positive for CK18 and Alb markers. The hepatocyte-like cells functional aspects were observed by glycogen storage. Though a heterogeneous cell hepatic lineage was confirmed, mouse placental scaffolds shown a useful model to support recellularization with simultaneous differentiation into hepatic fate simulating phases of embryonic development. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-02-26 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://www.teses.usp.br/teses/disponiveis/10/10132/tde-31072018-161001/ |
url |
http://www.teses.usp.br/teses/disponiveis/10/10132/tde-31072018-161001/ |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
|
dc.rights.driver.fl_str_mv |
Liberar o conteúdo para acesso público. info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Liberar o conteúdo para acesso público. |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.coverage.none.fl_str_mv |
|
dc.publisher.none.fl_str_mv |
Biblioteca Digitais de Teses e Dissertações da USP |
publisher.none.fl_str_mv |
Biblioteca Digitais de Teses e Dissertações da USP |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da USP instname:Universidade de São Paulo (USP) instacron:USP |
instname_str |
Universidade de São Paulo (USP) |
instacron_str |
USP |
institution |
USP |
reponame_str |
Biblioteca Digital de Teses e Dissertações da USP |
collection |
Biblioteca Digital de Teses e Dissertações da USP |
repository.name.fl_str_mv |
Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP) |
repository.mail.fl_str_mv |
virginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.br |
_version_ |
1815257514555473920 |