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O▪93 Generation of new diploid and triploid human embryonic stem cell lines
Abstracts - 6th International Symposium on Preimplantation Genetics 2005
O 92 PGD for HLA typing can be a valuable source for the establishment of new human embryonic stem cell lines Findikli N1, Kahraman S1, Akcin O1, Sertyel S1, Candan Z1, Karlikaya G1, Fiorentino F2 1Istanbul Memorial Hospital, ART and Reproductive Genetics Centre, R and D Laboratory, Istanbul, Turkey; 2‘GENOMA’Molecular Genetics Laboratory, Rome, Italy Objective: Human embryonic stem cells are pluripotent cells derived from the inner cell mass of human blastocysts. Here, we report the isolation, establishment and characterization human embryonic stem cell lines as well as their differentiation potential into variety of somatic cell types after preimplantation genetic diagnosis (PGD) for human leukocyte antigen (HLA) typing. Materials and methods: Thirty-one human blastocysts were donated for human embryonic stem cell research. Six were from a PGD cycle in combination with HLA typing (PGD– HLA). Human embryonic stem cell isolation was performed either by immunosurgery or by direct culture method. All cell lines were tested for their karyotype, surface antigens (SSEA4, TRA-1–60, TRA-1–81, OCT-4), and alkaline phosphatase expression as well as their in-vitro differentiation potential. Also, cell lines were cryopreserved by vitrification at certain passages. Results: Overall, seven human embryonic stem cell lines were established and of them, four were from blastocysts originating in PGD–HLA cycles. All four lines were found to display a typical stem cell-like cellular morphology and immunocytochemistry characteristics with normal karyotypes. They were also successfully cryopreserved/thawed and showed similar growth and cellular properties upon thawing. When induced to differentiate in vitro, these cells formed cellular structures containing spontaneously contracting areas, neuronal as well as endothelial-like structures indicating the potential to form three germ layers. Conclusions: Besides the financial and ethical limits imposed on human embryonic stem cell research, lower success rates are mostly due to low quality blastocysts donated for stem cell research. Our results can indicate that good quality embryos which are not suitable in PGD–HLA cycles can be valuable sources for the establishment of new human embryonic stem cell lines.
O 93 Generation of new diploid and triploid human embryonic stem cell lines Baharvand H, Ashtiani SK, Taee A, Masoumi M, Moradi S, Rezazadeh Valojerdi M, Eftekhari P Department of Stem Cells, Royan Institute, PO Box: 19395– 4644, Tehran, Iran Introduction: Human pluripotent embryonic stem (hES) cells have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of four hES cell lines on mouse embryonic fibroblast cells. Materials and methods: Stem cell lines were characterized
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by the morphology, passaging, and expression profiles of a number of genes, including TRA-1–60, TRA-1–81, alkaline phosphatase, OCT-4, NANOG, CXCR4, NODAL, LEFTYA, Thy-1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX-1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in-vitro conditions. Results: Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, two of them carried a triploid (69,XXY) karyotype. Conclusions: These results define establishment of diploid and triploid cells as new hES cell lines.
O 94 Improvement of the signs of Parkinson s disease in rats following transplantation of embryonic stem cells Fathi F1, Altiraihi T1, Mowahedin M1, Javad Mowla SJ2 1Department of Anatomy; 2Department of Genetics, Faculty of Basic Sciences, Tarbiat Modarres University, Ale Ahmad Highway, Tehran, Iran Introduction: Parkinson’s disease (PD) is a degenerative disease that is caused by the death of dopaminergic neurons, and the response to current treatments is variable. It is important to develop a model for evaluation of embryonic stem (ES) cells as an alternative model of treatment. Materials and methods: A model for PD was developed in rats. The ES cells were transplanted into three groups of rats: treated with retinoic acid (RA), not treated with RA, and transfected by BDNF gene. A fourth group received culture media. The ability of cells to improve Parkinson’s disease was evaluated, using the following method. The cells were labelled by BrdU. Histological and immunohistochemical evaluations were performed on days 5 and 15, and behavioural evaluation was carried out in week 18 after transplantation. On day 15, histological evaluation by haematoxylin and eosin and cresyl violet staining indicated that transplanted cells differentiated to neural cells and integrated into host tissue. Examination of transplanted cells by electron microscopy showed neurons and oligodendrocytes. The immunohistochemical result was positive for tyrosine hydroxylase, which is an important marker for dopaminergic neurons, and confirmed differentiation of transplanted cells to dopaminergic neurons. On day 5, ES cells labelled with BrdU and expressed SSEA1 antigen were detected in the transplantation area. Results: Histological and immunohistochemical investigation confirmed by behavioural tests showed improved in groups treated by RA, not treated by RA and transfected by BDNF gene, in comparison with the control group. Conclusions: The results indicate that CCE ES cells are an appropriate cell line for transfection and differentiation to dopaminergic neurons, which can be used for research based on cell and gene therapy.
O 92 PGD for HLA typing can be a valuable source for the establishment of new human embryonic stem cell lines Findikli N1, Kahraman S1, Akcin O1, Sertyel S1, Candan Z1, Karlikaya G1, Fiorentino F2 1Istanbul Memorial Hospital, ART and Reproductive Genetics Centre, R and D Laboratory, Istanbul, Turkey; 2‘GENOMA’Molecular Genetics Laboratory, Rome, Italy Objective: Human embryonic stem cells are pluripotent cells derived from the inner cell mass of human blastocysts. Here, we report the isolation, establishment and characterization human embryonic stem cell lines as well as their differentiation potential into variety of somatic cell types after preimplantation genetic diagnosis (PGD) for human leukocyte antigen (HLA) typing. Materials and methods: Thirty-one human blastocysts were donated for human embryonic stem cell research. Six were from a PGD cycle in combination with HLA typing (PGD– HLA). Human embryonic stem cell isolation was performed either by immunosurgery or by direct culture method. All cell lines were tested for their karyotype, surface antigens (SSEA4, TRA-1–60, TRA-1–81, OCT-4), and alkaline phosphatase expression as well as their in-vitro differentiation potential. Also, cell lines were cryopreserved by vitrification at certain passages. Results: Overall, seven human embryonic stem cell lines were established and of them, four were from blastocysts originating in PGD–HLA cycles. All four lines were found to display a typical stem cell-like cellular morphology and immunocytochemistry characteristics with normal karyotypes. They were also successfully cryopreserved/thawed and showed similar growth and cellular properties upon thawing. When induced to differentiate in vitro, these cells formed cellular structures containing spontaneously contracting areas, neuronal as well as endothelial-like structures indicating the potential to form three germ layers. Conclusions: Besides the financial and ethical limits imposed on human embryonic stem cell research, lower success rates are mostly due to low quality blastocysts donated for stem cell research. Our results can indicate that good quality embryos which are not suitable in PGD–HLA cycles can be valuable sources for the establishment of new human embryonic stem cell lines.
O 93 Generation of new diploid and triploid human embryonic stem cell lines Baharvand H, Ashtiani SK, Taee A, Masoumi M, Moradi S, Rezazadeh Valojerdi M, Eftekhari P Department of Stem Cells, Royan Institute, PO Box: 19395– 4644, Tehran, Iran Introduction: Human pluripotent embryonic stem (hES) cells have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of four hES cell lines on mouse embryonic fibroblast cells. Materials and methods: Stem cell lines were characterized
30
by the morphology, passaging, and expression profiles of a number of genes, including TRA-1–60, TRA-1–81, alkaline phosphatase, OCT-4, NANOG, CXCR4, NODAL, LEFTYA, Thy-1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX-1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in-vitro conditions. Results: Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, two of them carried a triploid (69,XXY) karyotype. Conclusions: These results define establishment of diploid and triploid cells as new hES cell lines.
O 94 Improvement of the signs of Parkinson s disease in rats following transplantation of embryonic stem cells Fathi F1, Altiraihi T1, Mowahedin M1, Javad Mowla SJ2 1Department of Anatomy; 2Department of Genetics, Faculty of Basic Sciences, Tarbiat Modarres University, Ale Ahmad Highway, Tehran, Iran Introduction: Parkinson’s disease (PD) is a degenerative disease that is caused by the death of dopaminergic neurons, and the response to current treatments is variable. It is important to develop a model for evaluation of embryonic stem (ES) cells as an alternative model of treatment. Materials and methods: A model for PD was developed in rats. The ES cells were transplanted into three groups of rats: treated with retinoic acid (RA), not treated with RA, and transfected by BDNF gene. A fourth group received culture media. The ability of cells to improve Parkinson’s disease was evaluated, using the following method. The cells were labelled by BrdU. Histological and immunohistochemical evaluations were performed on days 5 and 15, and behavioural evaluation was carried out in week 18 after transplantation. On day 15, histological evaluation by haematoxylin and eosin and cresyl violet staining indicated that transplanted cells differentiated to neural cells and integrated into host tissue. Examination of transplanted cells by electron microscopy showed neurons and oligodendrocytes. The immunohistochemical result was positive for tyrosine hydroxylase, which is an important marker for dopaminergic neurons, and confirmed differentiation of transplanted cells to dopaminergic neurons. On day 5, ES cells labelled with BrdU and expressed SSEA1 antigen were detected in the transplantation area. Results: Histological and immunohistochemical investigation confirmed by behavioural tests showed improved in groups treated by RA, not treated by RA and transfected by BDNF gene, in comparison with the control group. Conclusions: The results indicate that CCE ES cells are an appropriate cell line for transfection and differentiation to dopaminergic neurons, which can be used for research based on cell and gene therapy.