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Mesenchymal stem cell engineering by ARCA analog-capped mRNA

We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for in vitro mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2)...

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Autores principales: Andrzejewska, Anna, Grzela, Renata, Stankiewicz-Drogon, Anna, Rogujski, Piotr, Nagaraj, Siranjeevi, Darzynkiewicz, Edward, Lukomska, Barbara, Janowski, Miroslaw
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10425852/
https://www.ncbi.nlm.nih.gov/pubmed/37588684
http://dx.doi.org/10.1016/j.omtn.2023.07.006
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author Andrzejewska, Anna
Grzela, Renata
Stankiewicz-Drogon, Anna
Rogujski, Piotr
Nagaraj, Siranjeevi
Darzynkiewicz, Edward
Lukomska, Barbara
Janowski, Miroslaw
author_facet Andrzejewska, Anna
Grzela, Renata
Stankiewicz-Drogon, Anna
Rogujski, Piotr
Nagaraj, Siranjeevi
Darzynkiewicz, Edward
Lukomska, Barbara
Janowski, Miroslaw
author_sort Andrzejewska, Anna
collection PubMed
description We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for in vitro mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2) mRNA purification method (spin column vs. high-performance liquid chromatography [HPLC] column), and (3) mRNA capping (ARCA vs. β-S-ARCA D1 and β-S-ARCA D2). Dependent variables included protein production based on mRNA template (measured by the bioluminescence of reporter gene luciferase over hours), MSC metabolic activity corresponding with their wellbeing measured by CCK-8 over days, and endogenous expression of genes by RT-qPCR related to innate intracellular immune response and decapping at two time points: days 2 and 5. We have found that Lipofectamine 2000 outperforms TransIT, and used it throughout the study. Then, we showed that mRNA must be purified by HPLC to be relatively neutral to MSCs in terms of metabolic activity and endogenous protein production. Ultimately, we demonstrated that β-S-ARCA D1 enables higher protein production but at the cost of lower MSC metabolic activity, with no impact on RT-qPCR results. Thus Lipofectamine 2000-based in vitro transfection of HPLC-purified and ARCA- or β-S-ARCA D1-capped mRNA is optimal for MSC engineering.
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spelling pubmed-104258522023-08-16 Mesenchymal stem cell engineering by ARCA analog-capped mRNA Andrzejewska, Anna Grzela, Renata Stankiewicz-Drogon, Anna Rogujski, Piotr Nagaraj, Siranjeevi Darzynkiewicz, Edward Lukomska, Barbara Janowski, Miroslaw Mol Ther Nucleic Acids Original Article We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for in vitro mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2) mRNA purification method (spin column vs. high-performance liquid chromatography [HPLC] column), and (3) mRNA capping (ARCA vs. β-S-ARCA D1 and β-S-ARCA D2). Dependent variables included protein production based on mRNA template (measured by the bioluminescence of reporter gene luciferase over hours), MSC metabolic activity corresponding with their wellbeing measured by CCK-8 over days, and endogenous expression of genes by RT-qPCR related to innate intracellular immune response and decapping at two time points: days 2 and 5. We have found that Lipofectamine 2000 outperforms TransIT, and used it throughout the study. Then, we showed that mRNA must be purified by HPLC to be relatively neutral to MSCs in terms of metabolic activity and endogenous protein production. Ultimately, we demonstrated that β-S-ARCA D1 enables higher protein production but at the cost of lower MSC metabolic activity, with no impact on RT-qPCR results. Thus Lipofectamine 2000-based in vitro transfection of HPLC-purified and ARCA- or β-S-ARCA D1-capped mRNA is optimal for MSC engineering. American Society of Gene & Cell Therapy 2023-07-17 /pmc/articles/PMC10425852/ /pubmed/37588684 http://dx.doi.org/10.1016/j.omtn.2023.07.006 Text en © 2023 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original Article
Andrzejewska, Anna
Grzela, Renata
Stankiewicz-Drogon, Anna
Rogujski, Piotr
Nagaraj, Siranjeevi
Darzynkiewicz, Edward
Lukomska, Barbara
Janowski, Miroslaw
Mesenchymal stem cell engineering by ARCA analog-capped mRNA
title Mesenchymal stem cell engineering by ARCA analog-capped mRNA
title_full Mesenchymal stem cell engineering by ARCA analog-capped mRNA
title_fullStr Mesenchymal stem cell engineering by ARCA analog-capped mRNA
title_full_unstemmed Mesenchymal stem cell engineering by ARCA analog-capped mRNA
title_short Mesenchymal stem cell engineering by ARCA analog-capped mRNA
title_sort mesenchymal stem cell engineering by arca analog-capped mrna
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10425852/
https://www.ncbi.nlm.nih.gov/pubmed/37588684
http://dx.doi.org/10.1016/j.omtn.2023.07.006
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