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Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system

Previous studies have demonstrated that somatic cells fused with pluripotent stem cells can be reprogrammed on the basis of reprogramming factors acquired from the latter. However, fusion-reprogrammed cells are deemed unsuitable for therapeutic applications mainly because conventional fusion techniq...

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Autores principales: Okanojo, Masahiro, Okeyo, Kennedy O., Hanzawa, Hiroko, Kurosawa, Osamu, Oana, Hidehiro, Takeda, Shizu, Washizu, Masao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: AIP Publishing LLC 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614026/
https://www.ncbi.nlm.nih.gov/pubmed/31312284
http://dx.doi.org/10.1063/1.5098829
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author Okanojo, Masahiro
Okeyo, Kennedy O.
Hanzawa, Hiroko
Kurosawa, Osamu
Oana, Hidehiro
Takeda, Shizu
Washizu, Masao
author_facet Okanojo, Masahiro
Okeyo, Kennedy O.
Hanzawa, Hiroko
Kurosawa, Osamu
Oana, Hidehiro
Takeda, Shizu
Washizu, Masao
author_sort Okanojo, Masahiro
collection PubMed
description Previous studies have demonstrated that somatic cells fused with pluripotent stem cells can be reprogrammed on the basis of reprogramming factors acquired from the latter. However, fusion-reprogrammed cells are deemed unsuitable for therapeutic applications mainly because conventional fusion techniques often yield tetraploid fusants that contain exogenous genes acquired from the fusion partners. Here, we present a novel cell–cell topological reconnection technique and demonstrate its application to nuclear transplantation between a somatic cell and a stem cell without nuclei mixing. As a proof of concept, a microfluidic fusion chip embodied with a microslit (4 μm in width) to prevent nuclei mixing was developed and used to perform one-to-one electrofusion of a target somatic cell (Jurkat cell) with an induced pluripotent stem (iPS) cell. To extract its cytoplasm, the target cell was first topologically connected to a sacrificial iPS cell by electrofusion via a microslit, followed by shear flow removal of the latter to obtain a cytoplasm-depleted nucleus of the target cell. Then, to replace the lost cytoplasm, topological reconnection to a second iPS cell was performed similarly by electrofusion, followed by shear flow separation of the target cell to enable it acquire most of the iPS cytoplasm, but without nuclei mixing. Microscopic observation of target cells harvested and cultured post hoc in a microwell confirmed that they manifested cell division. Taken together, these results demonstrate the potential application of the cell–cell topological reconnection technique to somatic cell nuclear transplantation for the generation of autologous pluripotent stem cells.
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spelling pubmed-66140262019-07-16 Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system Okanojo, Masahiro Okeyo, Kennedy O. Hanzawa, Hiroko Kurosawa, Osamu Oana, Hidehiro Takeda, Shizu Washizu, Masao Biomicrofluidics Regular Articles Previous studies have demonstrated that somatic cells fused with pluripotent stem cells can be reprogrammed on the basis of reprogramming factors acquired from the latter. However, fusion-reprogrammed cells are deemed unsuitable for therapeutic applications mainly because conventional fusion techniques often yield tetraploid fusants that contain exogenous genes acquired from the fusion partners. Here, we present a novel cell–cell topological reconnection technique and demonstrate its application to nuclear transplantation between a somatic cell and a stem cell without nuclei mixing. As a proof of concept, a microfluidic fusion chip embodied with a microslit (4 μm in width) to prevent nuclei mixing was developed and used to perform one-to-one electrofusion of a target somatic cell (Jurkat cell) with an induced pluripotent stem (iPS) cell. To extract its cytoplasm, the target cell was first topologically connected to a sacrificial iPS cell by electrofusion via a microslit, followed by shear flow removal of the latter to obtain a cytoplasm-depleted nucleus of the target cell. Then, to replace the lost cytoplasm, topological reconnection to a second iPS cell was performed similarly by electrofusion, followed by shear flow separation of the target cell to enable it acquire most of the iPS cytoplasm, but without nuclei mixing. Microscopic observation of target cells harvested and cultured post hoc in a microwell confirmed that they manifested cell division. Taken together, these results demonstrate the potential application of the cell–cell topological reconnection technique to somatic cell nuclear transplantation for the generation of autologous pluripotent stem cells. AIP Publishing LLC 2019-06-10 /pmc/articles/PMC6614026/ /pubmed/31312284 http://dx.doi.org/10.1063/1.5098829 Text en © 2019 Author(s). 1932-1058/2019/13(3)/034115/12 All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Regular Articles
Okanojo, Masahiro
Okeyo, Kennedy O.
Hanzawa, Hiroko
Kurosawa, Osamu
Oana, Hidehiro
Takeda, Shizu
Washizu, Masao
Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system
title Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system
title_full Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system
title_fullStr Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system
title_full_unstemmed Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system
title_short Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system
title_sort nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system
topic Regular Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614026/
https://www.ncbi.nlm.nih.gov/pubmed/31312284
http://dx.doi.org/10.1063/1.5098829
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