The Experts below are selected from a list of 267 Experts worldwide ranked by ideXlab platform
Gianpiero D. Palermo - One of the best experts on this subject based on the ideXlab platform.
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Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model
Journal of Translational Medicine, 2006Co-Authors: N. Tanaka, Takumi Takeuchi, Queenie V. Neri, Eric Scott Sills, Gianpiero D. PalermoAbstract:Background To evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional Immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation.
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Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model
Journal of Translational Medicine, 2006Co-Authors: N. Tanaka, Takumi Takeuchi, Queenie V. Neri, Eric Scott Sills, Gianpiero D. PalermoAbstract:Background To evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional Immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation. Methods Intact blastocysts or isolated ICMs generated in a standard mouse strain were plated in medium with or without serum to compare ESC harvesting efficiency. ESC derivation was also undertaken in a feeder cell-free culture system. Results Although ICM growth and dissociation was comparable irrespective of the media components, an enhanced ESC harvest was observed in our serum-free medium ( p < 0.01). ESC harvest rate was not affected by ICM isolation technique but was attenuated in the feeder cell-free group. Conclusion Achieving successful techniques for human ESC research is fundamentally dependent on preliminary work using experimental animals. In this study, all experimentally developed ESC lines manifested similar features to ESCs obtained from intact blastocysts in standard culture. Cell/sera free murine ESC harvest and propagation are feasible procedures for an embryology laboratory and await refinements for translation to human medical research.
Mayuko Kurome - One of the best experts on this subject based on the ideXlab platform.
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targeting αgal epitopes for multi species embryo Immunosurgery
Reproduction Fertility and Development, 2019Co-Authors: Mayuko Kurome, Andrea Baehr, Kilian Simmet, Eva Maria Jemiller, Stefanie Egerer, Maik Dahlhoff, Valeri Zakhartchenko, Hiroshi NagashimaAbstract:Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE-cell epitopes, have been used as the antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyperacute rejection of pig-to-primate xenotransplants. Since GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs could be used as a novel antibody source for multi-species embryo Immunosurgery. Mouse, rabbit, pig and cattle blastocysts were used for the experiment. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all species tested. GTKO pig serum contained sufficient anti-αGal antibodies to induce complement-mediated lysis of TE cells in blastocysts from all species investigated. Intact ICMs could be successfully recovered and the majority showed the desired level of purity. Our study demonstrates that GTKO pig serum is a reliable and effective source of antibodies targeting the αGal epitopes of TE cells for multi-species embryo Immunosurgery.
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202 targeting galactosyl α 1 3 galactose αgal epitopes for multi species embryo Immunosurgery
Reproduction Fertility and Development, 2018Co-Authors: Mayuko Kurome, Andrea Baehr, Kilian Simmet, Eva Maria Jemiller, Maik Dahlhoff, Valeri Zakhartchenko, Barbara Kessler, Nikolai Klymiuk, E WolfAbstract:Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE cell epitopes, have been used as antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyper-acute rejection of pig-to-primate xenotransplants. Because GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they are expected to produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs can be used as a novel antibody source for embryo Immunosurgery. First, the presence of αGal epitopes in mouse (E3.5), rabbit (Day 4), pig (Day 6–7), and bovine (Day 7–8) blastocysts was examined by staining with fluorescein isothiocyanate (FITC)-conjugated BSI-B4 lectin (Sigma, St. Louis, MO, USA) that binds αGal. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all examined species. Next, pig blastocysts were incubated with a medium containing GTKO pig serum. Swollen TE cells were observed in some of the blastocysts already after 2 min and, after 10 min, almost all TE cells of these blastocysts were completely destroyed. No lysis was recorded when the same experiment was done with wild-type pig serum, suggesting the presence of sufficient quantities of anti-αGal antibodies in GTKO serum to coat the TE cells and induce their complement-mediated lysis. Finally, GTKO serum was systematically tested for Immunosurgery. Zona-free blastocysts of the species mentioned above were incubated with heat-inactivated GTKO pig serum for 1 h at 38°C. After washing, the blastocysts were labelled with Hoechst 33342 and TE was stained with FITC-conjugated concanavalin A (ConA) to distinguish the ICM from TE cells. Eventually, the blastocysts were individually incubated in complement solution for 30 to 40 min. Complement-mediated lysis of TE cells was efficiently induced in mouse, rabbit, pig, and bovine blastocysts (10/10, 7/7, 10/10, and 5/6, respectively), and intact ICM were successfully recovered from all species (100, 100, 60, and 80%, respectively). Double fluorescent staining with Hoechst 33342 and ConA clearly showed that the majority of isolated ICM was not contaminated with TE cells. Our study demonstrates that GTKO pig serum is a reliable source of antibodies targeting the αGal epitope of TE cells. Major advantages of using GTKO serum for embryo Immunosurgery are (1) that it can be produced easily in large batches, thus reducing experimental variation; and (2) that it reacts with a large number of different species, except for humans, apes, and old world monkeys that lack αGal epitopes. Interesting applications include the preparation of TE and ICM for transcriptome profiling or chimeric embryo complementation experiments. This work is supported by the German Research Council (TR-CRC 127).
N. Tanaka - One of the best experts on this subject based on the ideXlab platform.
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Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model
Journal of Translational Medicine, 2006Co-Authors: N. Tanaka, Takumi Takeuchi, Queenie V. Neri, Eric Scott Sills, Gianpiero D. PalermoAbstract:Background To evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional Immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation.
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Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model
Journal of Translational Medicine, 2006Co-Authors: N. Tanaka, Takumi Takeuchi, Queenie V. Neri, Eric Scott Sills, Gianpiero D. PalermoAbstract:Background To evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional Immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation. Methods Intact blastocysts or isolated ICMs generated in a standard mouse strain were plated in medium with or without serum to compare ESC harvesting efficiency. ESC derivation was also undertaken in a feeder cell-free culture system. Results Although ICM growth and dissociation was comparable irrespective of the media components, an enhanced ESC harvest was observed in our serum-free medium ( p < 0.01). ESC harvest rate was not affected by ICM isolation technique but was attenuated in the feeder cell-free group. Conclusion Achieving successful techniques for human ESC research is fundamentally dependent on preliminary work using experimental animals. In this study, all experimentally developed ESC lines manifested similar features to ESCs obtained from intact blastocysts in standard culture. Cell/sera free murine ESC harvest and propagation are feasible procedures for an embryology laboratory and await refinements for translation to human medical research.
Andrea Baehr - One of the best experts on this subject based on the ideXlab platform.
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targeting αgal epitopes for multi species embryo Immunosurgery
Reproduction Fertility and Development, 2019Co-Authors: Mayuko Kurome, Andrea Baehr, Kilian Simmet, Eva Maria Jemiller, Stefanie Egerer, Maik Dahlhoff, Valeri Zakhartchenko, Hiroshi NagashimaAbstract:Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE-cell epitopes, have been used as the antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyperacute rejection of pig-to-primate xenotransplants. Since GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs could be used as a novel antibody source for multi-species embryo Immunosurgery. Mouse, rabbit, pig and cattle blastocysts were used for the experiment. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all species tested. GTKO pig serum contained sufficient anti-αGal antibodies to induce complement-mediated lysis of TE cells in blastocysts from all species investigated. Intact ICMs could be successfully recovered and the majority showed the desired level of purity. Our study demonstrates that GTKO pig serum is a reliable and effective source of antibodies targeting the αGal epitopes of TE cells for multi-species embryo Immunosurgery.
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202 targeting galactosyl α 1 3 galactose αgal epitopes for multi species embryo Immunosurgery
Reproduction Fertility and Development, 2018Co-Authors: Mayuko Kurome, Andrea Baehr, Kilian Simmet, Eva Maria Jemiller, Maik Dahlhoff, Valeri Zakhartchenko, Barbara Kessler, Nikolai Klymiuk, E WolfAbstract:Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE cell epitopes, have been used as antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyper-acute rejection of pig-to-primate xenotransplants. Because GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they are expected to produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs can be used as a novel antibody source for embryo Immunosurgery. First, the presence of αGal epitopes in mouse (E3.5), rabbit (Day 4), pig (Day 6–7), and bovine (Day 7–8) blastocysts was examined by staining with fluorescein isothiocyanate (FITC)-conjugated BSI-B4 lectin (Sigma, St. Louis, MO, USA) that binds αGal. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all examined species. Next, pig blastocysts were incubated with a medium containing GTKO pig serum. Swollen TE cells were observed in some of the blastocysts already after 2 min and, after 10 min, almost all TE cells of these blastocysts were completely destroyed. No lysis was recorded when the same experiment was done with wild-type pig serum, suggesting the presence of sufficient quantities of anti-αGal antibodies in GTKO serum to coat the TE cells and induce their complement-mediated lysis. Finally, GTKO serum was systematically tested for Immunosurgery. Zona-free blastocysts of the species mentioned above were incubated with heat-inactivated GTKO pig serum for 1 h at 38°C. After washing, the blastocysts were labelled with Hoechst 33342 and TE was stained with FITC-conjugated concanavalin A (ConA) to distinguish the ICM from TE cells. Eventually, the blastocysts were individually incubated in complement solution for 30 to 40 min. Complement-mediated lysis of TE cells was efficiently induced in mouse, rabbit, pig, and bovine blastocysts (10/10, 7/7, 10/10, and 5/6, respectively), and intact ICM were successfully recovered from all species (100, 100, 60, and 80%, respectively). Double fluorescent staining with Hoechst 33342 and ConA clearly showed that the majority of isolated ICM was not contaminated with TE cells. Our study demonstrates that GTKO pig serum is a reliable source of antibodies targeting the αGal epitope of TE cells. Major advantages of using GTKO serum for embryo Immunosurgery are (1) that it can be produced easily in large batches, thus reducing experimental variation; and (2) that it reacts with a large number of different species, except for humans, apes, and old world monkeys that lack αGal epitopes. Interesting applications include the preparation of TE and ICM for transcriptome profiling or chimeric embryo complementation experiments. This work is supported by the German Research Council (TR-CRC 127).
Hyun Soo Yoon - One of the best experts on this subject based on the ideXlab platform.
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establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum free condition
Biology of Reproduction, 2005Co-Authors: Jong Hyuk Park, Hyun Soo YoonAbstract:Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cellreplacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by Immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.
