Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells

Autores
Locatelli, Paola; Olea, Fernanda Daniela; Hnatiuk, Anna; Sepúlveda, Diana Elizabeth; Pérez Sáez, Juan Manuel; Argüello, Rafael; Crottogini, Alberto Jose
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
BACKGROUND AIMS: Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability. METHODS: Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass. RESULTS: Lipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection. CONCLUSIONS: Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.
Fil: Locatelli, Paola. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Olea, Fernanda Daniela. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Hnatiuk, Anna. Universidad Favaloro; Argentina
Fil: Sepúlveda, Diana Elizabeth. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Pérez Sáez, Juan Manuel. Bio Sidus S.A; Argentina
Fil: Argüello, Rafael. Universidad Favaloro; Argentina
Fil: Crottogini, Alberto Jose. Universidad Favaloro; Argentina
Materia
Mesenchymal Stromal Cell
Plasmids
Transfection Efficiency
Sheep
Vegf
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/22913
Acceder
id CONICETDig_668981461043964d2b05dcee6d241a28
oai_identifier_str oai:ri.conicet.gov.ar:11336/22913
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cellsLocatelli, PaolaOlea, Fernanda DanielaHnatiuk, AnnaSepúlveda, Diana ElizabethPérez Sáez, Juan ManuelArgüello, RafaelCrottogini, Alberto JoseMesenchymal Stromal CellPlasmidsTransfection EfficiencySheepVegfhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1BACKGROUND AIMS: Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability. METHODS: Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass. RESULTS: Lipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection. CONCLUSIONS: Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.Fil: Locatelli, Paola. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olea, Fernanda Daniela. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hnatiuk, Anna. Universidad Favaloro; ArgentinaFil: Sepúlveda, Diana Elizabeth. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pérez Sáez, Juan Manuel. Bio Sidus S.A; ArgentinaFil: Argüello, Rafael. Universidad Favaloro; ArgentinaFil: Crottogini, Alberto Jose. Universidad Favaloro; ArgentinaElsevier2013-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/22913Locatelli, Paola; Olea, Fernanda Daniela; Hnatiuk, Anna; Sepúlveda, Diana Elizabeth; Pérez Sáez, Juan Manuel; et al.; Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells; Elsevier; Cytotherapy; 15; 2; 1-2013; 163-1701465-3249CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S1465324912000333info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jcyt.2012.11.004info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:08:01Zoai:ri.conicet.gov.ar:11336/22913instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:08:02.132CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
title Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
spellingShingle Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
Locatelli, Paola
Mesenchymal Stromal Cell
Plasmids
Transfection Efficiency
Sheep
Vegf
title_short Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
title_full Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
title_fullStr Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
title_full_unstemmed Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
title_sort Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells
dc.creator.none.fl_str_mv Locatelli, Paola
Olea, Fernanda Daniela
Hnatiuk, Anna
Sepúlveda, Diana Elizabeth
Pérez Sáez, Juan Manuel
Argüello, Rafael
Crottogini, Alberto Jose
author Locatelli, Paola
author_facet Locatelli, Paola
Olea, Fernanda Daniela
Hnatiuk, Anna
Sepúlveda, Diana Elizabeth
Pérez Sáez, Juan Manuel
Argüello, Rafael
Crottogini, Alberto Jose
author_role author
author2 Olea, Fernanda Daniela
Hnatiuk, Anna
Sepúlveda, Diana Elizabeth
Pérez Sáez, Juan Manuel
Argüello, Rafael
Crottogini, Alberto Jose
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Mesenchymal Stromal Cell
Plasmids
Transfection Efficiency
Sheep
Vegf
topic Mesenchymal Stromal Cell
Plasmids
Transfection Efficiency
Sheep
Vegf
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv BACKGROUND AIMS: Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability. METHODS: Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass. RESULTS: Lipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection. CONCLUSIONS: Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.
Fil: Locatelli, Paola. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Olea, Fernanda Daniela. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Hnatiuk, Anna. Universidad Favaloro; Argentina
Fil: Sepúlveda, Diana Elizabeth. Universidad Favaloro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Pérez Sáez, Juan Manuel. Bio Sidus S.A; Argentina
Fil: Argüello, Rafael. Universidad Favaloro; Argentina
Fil: Crottogini, Alberto Jose. Universidad Favaloro; Argentina
description BACKGROUND AIMS: Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability. METHODS: Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass. RESULTS: Lipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection. CONCLUSIONS: Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.
publishDate 2013
dc.date.none.fl_str_mv 2013-01
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/22913
Locatelli, Paola; Olea, Fernanda Daniela; Hnatiuk, Anna; Sepúlveda, Diana Elizabeth; Pérez Sáez, Juan Manuel; et al.; Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells; Elsevier; Cytotherapy; 15; 2; 1-2013; 163-170
1465-3249
CONICET Digital
CONICET
url http://hdl.handle.net/11336/22913
identifier_str_mv Locatelli, Paola; Olea, Fernanda Daniela; Hnatiuk, Anna; Sepúlveda, Diana Elizabeth; Pérez Sáez, Juan Manuel; et al.; Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells; Elsevier; Cytotherapy; 15; 2; 1-2013; 163-170
1465-3249
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S1465324912000333
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jcyt.2012.11.004
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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