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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
AIP Publishing LLC
2019
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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. |
format | Online Article Text |
id | pubmed-6614026 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | AIP Publishing LLC |
record_format | MEDLINE/PubMed |
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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