Manufacturing of mesenchymal stromal cell (MSC)-based therapies for regenerative medicine requires the use of suitable supply of growth factors that enhance proliferation, cell stability and potency during cell expansion. Human blood derivatives such as human platelet lysate (hPL) have emerged as a feasible alternative for cell growth supplement. Nevertheless, composition and functional characterization of hPL in the context of cell manufacturing is still under investigation, particularly regarding the content and function of pro-survival and pro-regenerative factors. We performed comparative analyses of hPL, human serum (hS) and fetal bovine serum (FBS) stability and potency to support Wharton’s jelly (WJ) MSC production. We demonstrated that hPL displayed low inter-batch variation and unique secretome profile that was not present in hS and FBS. Importantly, hPL-derived factors including PDGF family, EGF, TGF-alpha, angiogenin and RANTES were actively taken up by WJ-MSC to support efficient expansion. Moreover, hPL but not hS or FBS induced secretion of osteoprotegerin, HGF, IL-6 and GRO-alpha by WJ-MSC during the expansion phase. Thus, hPL is a suitable source of factors supporting viability, stability and potency of WJ-MSC and therefore constitutes an essential raw material that in combination with WJ-MSC introduces a great opportunity for the generation of potent regenerative medicine products.

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.

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 2 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.