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Establishment of the optimal mRNA electroporation protocol for human skin-derived mesenchymal stem cells. / Neuckermans, Jessie; Branson, Steven; Vanhaecke, Tamara; De Kock, Joery.

2017. Poster session presented at PhD Day, Jette, Belgium.

Research output: Unpublished contribution to conferencePoster

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@conference{78563095242d4e11b638f33f186ebfc1,
title = "Establishment of the optimal mRNA electroporation protocol for human skin-derived mesenchymal stem cells.",
abstract = "Background: Stem cell therapy using so-called human skin-derived mesenchymal stem cells (sMSC) could offer a possible alternative to orthotopic liver transplantation for the treatment of inborn errors of liver metabolism. However the success of stem cell therapy in general for the treatment of liver disease is currently limited, because over 70{\%} of the transplanted cells are cleared within the first 24h to 48h. We hypothesize that transient modification of sMSCs with synthetic mRNAs coding for liver-specific cell adhesion molecules could improve their liver engraftment.Aim: The aim of this first study is to select the most suitable electroporation protocol for sMSCs, by using synthetic mRNA coding for Green Fluorescent Protein (GFP).Methods: Two electroporation protocols are compared: (1) exponential decay (ED) and (2) square wave (SW) pulse. The cell viability and the transfection efficiency is examined 24, 48, 72 and 168 hours after electroporation. Both parameters are evaluated by flow cytometry and immunocytochemistry.Results: We found that the cell viability is slightly decreased using the SW method compared to the ED method. However, the GFP expression in sMSCs per cell is approximately twice as high using the SW protocol, while the percentage of GFP positive cells (± 90{\%}) remains just about the same. These results were quantified using flow cytometry and confirmed by immunocytochemistry.Conclusion: Altogether, the SW method is considered to be the most optimal mRNA electroporation method for sMSCs as only a minor decrease in cell viability could be observed, but a significantly two-fold higher protein expression is obtained. As a consequence, the SW method will either lead to more protein expression per cell or will allow to use less mRNA in order to achieve the same protein expression as the ED protocol. This is particularly interesting when simultaneous electroporation of several different synthetic mRNA’s is required.",
author = "Jessie Neuckermans and Steven Branson and Tamara Vanhaecke and {De Kock}, Joery",
year = "2017",
language = "English",
note = "PhD Day ; Conference date: 28-03-2017 Through 28-03-2017",
url = "https://my.vub.ac.be/nieuws/2017/02/23/phd-day-march-28-2017-campus-jette, https://my.vub.ac.be/nieuws/2017/02/23/phd-day-march-28-2017-campus-jette, https://my.vub.ac.be/nieuws/2017/02/23/phd-day-march-28-2017-campus-jette",

}

RIS

TY - CONF

T1 - Establishment of the optimal mRNA electroporation protocol for human skin-derived mesenchymal stem cells.

AU - Neuckermans, Jessie

AU - Branson, Steven

AU - Vanhaecke, Tamara

AU - De Kock, Joery

PY - 2017

Y1 - 2017

N2 - Background: Stem cell therapy using so-called human skin-derived mesenchymal stem cells (sMSC) could offer a possible alternative to orthotopic liver transplantation for the treatment of inborn errors of liver metabolism. However the success of stem cell therapy in general for the treatment of liver disease is currently limited, because over 70% of the transplanted cells are cleared within the first 24h to 48h. We hypothesize that transient modification of sMSCs with synthetic mRNAs coding for liver-specific cell adhesion molecules could improve their liver engraftment.Aim: The aim of this first study is to select the most suitable electroporation protocol for sMSCs, by using synthetic mRNA coding for Green Fluorescent Protein (GFP).Methods: Two electroporation protocols are compared: (1) exponential decay (ED) and (2) square wave (SW) pulse. The cell viability and the transfection efficiency is examined 24, 48, 72 and 168 hours after electroporation. Both parameters are evaluated by flow cytometry and immunocytochemistry.Results: We found that the cell viability is slightly decreased using the SW method compared to the ED method. However, the GFP expression in sMSCs per cell is approximately twice as high using the SW protocol, while the percentage of GFP positive cells (± 90%) remains just about the same. These results were quantified using flow cytometry and confirmed by immunocytochemistry.Conclusion: Altogether, the SW method is considered to be the most optimal mRNA electroporation method for sMSCs as only a minor decrease in cell viability could be observed, but a significantly two-fold higher protein expression is obtained. As a consequence, the SW method will either lead to more protein expression per cell or will allow to use less mRNA in order to achieve the same protein expression as the ED protocol. This is particularly interesting when simultaneous electroporation of several different synthetic mRNA’s is required.

AB - Background: Stem cell therapy using so-called human skin-derived mesenchymal stem cells (sMSC) could offer a possible alternative to orthotopic liver transplantation for the treatment of inborn errors of liver metabolism. However the success of stem cell therapy in general for the treatment of liver disease is currently limited, because over 70% of the transplanted cells are cleared within the first 24h to 48h. We hypothesize that transient modification of sMSCs with synthetic mRNAs coding for liver-specific cell adhesion molecules could improve their liver engraftment.Aim: The aim of this first study is to select the most suitable electroporation protocol for sMSCs, by using synthetic mRNA coding for Green Fluorescent Protein (GFP).Methods: Two electroporation protocols are compared: (1) exponential decay (ED) and (2) square wave (SW) pulse. The cell viability and the transfection efficiency is examined 24, 48, 72 and 168 hours after electroporation. Both parameters are evaluated by flow cytometry and immunocytochemistry.Results: We found that the cell viability is slightly decreased using the SW method compared to the ED method. However, the GFP expression in sMSCs per cell is approximately twice as high using the SW protocol, while the percentage of GFP positive cells (± 90%) remains just about the same. These results were quantified using flow cytometry and confirmed by immunocytochemistry.Conclusion: Altogether, the SW method is considered to be the most optimal mRNA electroporation method for sMSCs as only a minor decrease in cell viability could be observed, but a significantly two-fold higher protein expression is obtained. As a consequence, the SW method will either lead to more protein expression per cell or will allow to use less mRNA in order to achieve the same protein expression as the ED protocol. This is particularly interesting when simultaneous electroporation of several different synthetic mRNA’s is required.

M3 - Poster

ER -

ID: 36815693