Abstract
Background Wharton’s Jelly-derived mesenchymal stromal cells (WJ-MSCs) present several advantages over other sources of multipotent stem cells, not only because they are obtained from neonatal umbilical cord, which is considered a biological waste, but also display higher proliferation rate and low senescence at later passages compared to stromal cells obtained from other sources. In the field of tissue engineering, WJ-MSCs have a wide therapeutic potential, due to their multipotential capacity, which can be reinforced if cells are genetically modified to direct their differentiation towards a specific lineage; unfortunately, as primary cells, WJ-MSC are difficult to transfect. Therefore, the objective of the present work was to standardize a protocol for the transfection of WJ-MSCs using a cationic polymer. Such protocol is important for future developments that contemplate the genetic modification of WJ-MSCs for therapeutic purposes.
Methods In this work, WJ-MSCs were genetically modified using polyethylenimine (PEI) and a lentiviral plasmid that encodes for green fluorescent protein (pGFP). To achieve WJ-MSCs transfection, complexes between PEI and pGFP, varying its composition (N/P ratio), were evaluated and characterized by size, zeta potential and cytotoxicity. At the N/P ratio condition where the highest transfection efficiencies were obtained, immunophenotype, immunomodulation properties and multipotential capacity of WJ-MSCs were evaluated.
Results Here, we present the standardization of the transfection conditions of the WJ-MSCs in a monolayer culture with PEI. The concentrations of plasmid and PEI that have the best transfection efficiencies were established
Conclusions Transfection with PEI doesn’t affect immunophenotype, immunomodulatory properties and differentiation capacity of WJ-MSCs.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- Ag
- Specific surface antigen
- DAPI
- 4′,6-diamidino-2-phenylindole
- DLS
- Dynamic Light Scattering
- DMEM
- Dulbecco’s Modified Eagles Medium
- DMSO
- Dimethyl sulfoxide
- DW
- Distilled water
- FBS
- Fetal Bovine Serum
- GFP
- Green fluorescent protein
- HCl
- chloride acid
- HLA
- Human leukocyte antigen
- HLA-AB
- HLA class I molecules are present in all nucleated cells in human. This region includes HLA-A, -B, -C, -E, -F, -G, -H, -J, and HLA-X loci. HLA-A, HLA-B, and HLA-C loci are polymorphic and functional classical class I loci
- HLA-DR
- HLA class II molecules are located on B lymphocytes, macrophages, dendritic cells, endothelial cells, and active T cells. They consist of six different loci (HLA-DM, DN, DO, DP, DQ, and DR).
- hMSCs
- Human mesenchymal stromal cells
- ISCT
- International Society for Cell and Gene Therapy
- LB
- Luria Bertani
- MSCs
- Mesenchymal stem cells
- N/P ratio
- N correspond to the nitrogen in PEI and P refers to phosphate content in DNA.
- NADH
- Nicotinamide adenine dinucleotide exists in two forms: an oxidized and reduced form, abbreviated as NAD+ and NADH
- NADPH
- NADPH is the reduced form of NADP+ (Nicotinamide adenine dinucleotide phosphate)
- OD600nm
- Optic density at 600 nm
- PBMCs (A)
- Peripheral blood mononuclear cells activated
- PBMCs (A/A-WJ hMSCs)
- Peripheral blood mononuclear cells activated and co-cultured with mesenchymal stromal cells
- PBMCs (NA)
- Peripheral blood mononuclear cells and without activation
- PBMCs
- Peripheral blood mononuclear cells (PBMCs)
- PBS
- Phosphate Buffered Solution
- PEI
- Polyethylenimine
- pGFP
- Plasmid codifying for green fluorescent protein
- RFU
- Relative Fluorescence Units
- TAE
- Tris-acetate-EDTA
- TGF-β
- Transforming Growth Factor-β
- UC-hMSCs
- Umbilical cord human mesenchymal stromal cells
- WJ-MSCs
- Wharton’s Jelly-derived mesenchymal stromal cells
- WJ-MSCs (NT)
- Wharton’s Jelly-derived mesenchymal stromal cells non-transfected
- WJ-MSCs (T)
- Wharton’s Jelly-derived mesenchymal stromal cells transfected with polyethylenimine