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Efficient Non-Viral Gene Modification of Mesenchymal Stromal Cells from Umbilical Cord Wharton's Jelly with Polyethylenimine.

ABSTRACT: Mesenchymal stromal cells (MSC) derived from human umbilical cord Wharton's jelly (WJ) have a wide therapeutic potential in cell therapy and tissue engineering because of their multipotential capacity, which can be reinforced through gene therapy in order to modulate specific responses. However, reported methodologies to transfect WJ-MSC using cationic polymers are scarce. Here, WJ-MSC were transfected using 25 kDa branched- polyethylenimine (PEI) and a DNA plasmid encoding GFP. PEI/plasmid complexes were characterized to establish the best transfection efficiencies with lowest toxicity. Expression of MSC-related cell surface markers was evaluated. Likewise, immunomodulatory activity and multipotential capacity of transfected WJ-MSC were assessed by CD2/CD3/CD28-activated peripheral blood mononuclear cells (PBMC) cocultures and osteogenic and adipogenic differentiation assays, respectively. An association between cell number, PEI and DNA content, and transfection efficiency was observed. The highest transfection efficiency (15.3 ± 8.6%) at the lowest toxicity was achieved using 2 ng/?L DNA and 3.6 ng/?L PEI with 45,000 WJ-MSC in a 24-well plate format (200 ?L). Under these conditions, there was no significant difference between the expression of MSC-identity markers, inhibitory effect on CD3+ T lymphocytes proliferation and osteogenic/adipogenic differentiation ability of transfected WJ-MSC, as compared with non-transfected cells. These results suggest that the functional properties of WJ-MSC were not altered after optimized transfection with PEI.

SUBMITTER: Ramos-Murillo AI

PROVIDER: S-EPMC7559368 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Efficient Non-Viral Gene Modification of Mesenchymal Stromal Cells from Umbilical Cord Wharton's Jelly with Polyethylenimine.

Ramos-Murillo Ana Isabel AI Rodríguez Elizabeth E Beltrán Karl K Ricaurte Cristian C Camacho Bernardo B Salguero Gustavo G Godoy-Silva Rubén Darío RD

Pharmaceutics 20200922 9

Mesenchymal stromal cells (MSC) derived from human umbilical cord Wharton's jelly (WJ) have a wide therapeutic potential in cell therapy and tissue engineering because of their multipotential capacity, which can be reinforced through gene therapy in order to modulate specific responses. However, reported methodologies to transfect WJ-MSC using cationic polymers are scarce. Here, WJ-MSC were transfected using 25 kDa branched- polyethylenimine (PEI) and a DNA plasmid encoding GFP. PEI/plasmid comp ...[more]

PMID: 32971730

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Project description:Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a novel and promising strategy for tissue engineering because of their ability to differentiate into many cell types. We characterized the differentiation of WJ-MSCs into endometrial epithelial cell (EEC)-like and endometrial stromal cell (ESC)-like cells and assessed the effect of 17?-estradiol and 8-Br-cAMP on the differentiation system.WJ-MSCs were treated in two ways to differentiate into EEC-like and ESC-like cells respectively: cocultured with ESCs in control/differentiation medium (17?-estradiol, growth factors); and cultured in control/differentiation medium (8-Br-cAMP alone or 8-Br-cAMP plus 17?-estrogen and growth factors). Three signaling pathway inhibitors (SB203580, PD98059, H89) were used to investigate the mechanism of WJ-MSC differentiation into ESC-like cells. Immunofluorescence, western blot and flow cytometry analyses were used to analyze expression of epithelial markers and stromal cell markers. Enzyme-linked immunosorbent assays were used to test the production of secretory proteins associated with the differentiation of ESC-like cells.17?-estradiol at 1 ?M downregulated vimentin and CD13 and upregulated cytokeratin and CD9 proteins, promoting the differentiation of WJ-MSCs into EEC-like cells in the coculture system. 8-Br-cAMP at 0.5 mM upregulated vimentin and CD13 and downregulated CK and CD9, promoting the differentiation of WJ-MSCs into ESC-like cells. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) were upregulated and the protein kinase A (PKA) signaling pathway was activated, whereas extracellular signal-regulated (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were not affected.17?-estradiol at 1 ?M is a good inducer for facilitating the differentiation of WJ-MSCs into EEC-like cells. 8-Br-cAMP plus estrogen and growth factors can induce the differentiation of WJ-MSCs into ESC-like cells. During the differentiation of WJ-MSCs into ESC-like cells, PRL and IGFBP1 were upregulated by the treatment and the PKA signaling pathway was activated, whereas ERK1/2 and p38 MAPK were not affected. These findings suggest a promising approach to the treatment of endometrial damage and other endometrial diseases and suggest new applications for WJ-MSCs in clinical practice.

Dataset Information

Efficient Non-Viral Gene Modification of Mesenchymal Stromal Cells from Umbilical Cord Wharton's Jelly with Polyethylenimine.

Publications

Efficient Non-Viral Gene Modification of Mesenchymal Stromal Cells from Umbilical Cord Wharton's Jelly with Polyethylenimine.

Similar Datasets

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