Publicação
Optimization of the differentiation process of umbilical cord matrix mesenchymal stem cells into hepatocyte-like cells
| Resumo: | The few literature described protocols of hepatic differentiation of human umbilical cord matrix mesenchymal stem cells (ucmMSC) lead to populations of cells with less than satisfactory differentiation rates. In this study a hepatic differentiation protocol was firstly optimized in conventional monolayer (2D) cultures and further implemented in a 3D culture method, in order to achieve a homogenous population of functional hepatocyte-like cells (HLCs), as part of the creation of an alternative model for in vitro toxicology studies. UCXR, ucmMSC isolated by a proprietary method developed by ECBio S.A., were subjected to 4 differentiation protocols in 2D culture conditions being the most promising procedure applied to 3D culture method. The characterization of differentiated UCXR included the analysis of the expression of hepatic markers by quantitative real time reverse--‐transcription polymerase chain reaction (qRT-PCR) and immunofluorescence assays. Metabolic activity was assessed through ECOD and UGT activity assay, and also by glycogen accumulation and urea production. Undifferentiated UCXR, HepG2 and primary rat hepatocytes were used as controls. The HLCs that resulted from the optimized protocol showed hepatocyte-like morphology, expression of hepatic lineage markers, including ALB, CK18 and HNF4α as well as the underexpression of CK19. These also exhibited biotransformation activity (ECOD and UGT activities), and the ability to store glycogen and to produce urea. When transposed into 3D cultures, the optimized method induced the expression of hepatic markers CK18, HNF4α and ALB and higher urea production. In summary a more homogenous and functional population of HLCs, with hepatocyte expression pattern and metabolic activity at a superior level than HepG2 and in some aspects at the same activity level of rat primary hepatocytes, was successfully obtained, opening doors to the construction of a humanly-close metabolism and toxicology model for drug testing. |
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| Autores principais: | Silva, Alexandra Martins de Medeiros Vieira da |
| Assunto: | Diferenciação celular Células estaminais Cordão umbilical Hepatócitos Teses de mestrado - 2013 |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | The few literature described protocols of hepatic differentiation of human umbilical cord matrix mesenchymal stem cells (ucmMSC) lead to populations of cells with less than satisfactory differentiation rates. In this study a hepatic differentiation protocol was firstly optimized in conventional monolayer (2D) cultures and further implemented in a 3D culture method, in order to achieve a homogenous population of functional hepatocyte-like cells (HLCs), as part of the creation of an alternative model for in vitro toxicology studies. UCXR, ucmMSC isolated by a proprietary method developed by ECBio S.A., were subjected to 4 differentiation protocols in 2D culture conditions being the most promising procedure applied to 3D culture method. The characterization of differentiated UCXR included the analysis of the expression of hepatic markers by quantitative real time reverse--‐transcription polymerase chain reaction (qRT-PCR) and immunofluorescence assays. Metabolic activity was assessed through ECOD and UGT activity assay, and also by glycogen accumulation and urea production. Undifferentiated UCXR, HepG2 and primary rat hepatocytes were used as controls. The HLCs that resulted from the optimized protocol showed hepatocyte-like morphology, expression of hepatic lineage markers, including ALB, CK18 and HNF4α as well as the underexpression of CK19. These also exhibited biotransformation activity (ECOD and UGT activities), and the ability to store glycogen and to produce urea. When transposed into 3D cultures, the optimized method induced the expression of hepatic markers CK18, HNF4α and ALB and higher urea production. In summary a more homogenous and functional population of HLCs, with hepatocyte expression pattern and metabolic activity at a superior level than HepG2 and in some aspects at the same activity level of rat primary hepatocytes, was successfully obtained, opening doors to the construction of a humanly-close metabolism and toxicology model for drug testing. |
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