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/μL DNA and 3.6 ng/μL PEI with 45,000 WJ-MSC in a 24-well plate format (200 μL). 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.

In vitro assessments of nanoplexes of polyethylenimine-coated graphene oxide-plasmid through various cancer cell lines and primary mesenchymal stem cells

“…For instance, Ramos-Murillo et al reported 15.3 ± 8.6% transfection efficiency using bPEI 25 kDa in MSCs isolated from Umbilical Cord Wharton's Jelly. Interestingly, the viability of MSCs treated with PEI in their study was similarly reduced, comparable to our observations with the same concentration of PEI [32]. According to MTT assays in the present work, treatment with GO-PEI reduces the viability of MSC and Calu-3 cells by almost 25%, which are the most declining viability rates among the other studied cell types.…”

Development of a Gene-Activated Scaffold Incorporating Multifunctional Cell-Penetrating Peptides for pSDF-1α Delivery for Enhanced Angiogenesis in Tissue Engineering Applications

“…Interestingly, GET* N/P 8 nanoparticles elicited the highest transfection efficiency in MSCs in 2D, significantly more so than unmodified GET and GET* N/P 6, and was also higher than the GET* N/P 11 group. This contrasts with similar optimisation studies in MSCs, where increasing N/P ratio tends to correlate with increased transfection efficiency [58,59]. One potential hypothesis to explain this result is that the increased positive charge conferred on GET* N/P 11 particles as a result of an increased proportion of FLH to FLR causes a more pronounced 'proton-sponge' effect and subsequently less viable cells can be transfected.…”

Cryopreservation does not change the performance and characteristics of allogenic mesenchymal stem cells highly over-expressing a cytoplasmic therapeutic transgene for cancer treatment

“…As there is no genome integration, transgene expression can be reduced or lost post-cryopreservation [19]. Additionally, transfection reagents may compromise the integrity of cell membrane, raising concerns over the possibility of reduced cell viability during cryopreservation [20,21]. Metabolic burden imposed by significant exogenously expressed gene load can have detrimental effects [22], rendering the cells to be more vulnerable to the freezing and thawing process.…”