mRNA transfection – a novel, clinically applicable tool for the robust induction of transient transgene expression in human mesenchymal stem cells

• Autorzy: Nowakowski A. , Andrzejewska A. , Kozlowska H. , Walczak P. , Lukomska B. , Janowski M.
• Języki publikacji: EN

Abstrakty

EN

Adult bone marrow-derived mesenchymal stem cells (hMSCs) display a spectrum of functional properties. Transplantation of these cells improves the clinical outcome in experimental models of cerebral ischemia and spinal cord injury. Therapeutic effects have been reported in stroke after the systemic delivery of MSCs. A minimally invasive, intraarterial route is an attractive method for stem cell transplantation to the injured brain. However, MSCs lack the intrinsic mechanisms that enable homing of the cells to the area of infarction. Recent studies suggest that genetic manipulation can promote the forced expression of certain molecules responsible for adhesion and transendothelial migration of systemically delivered cells. It is anticipated that, for cell homing to the brain after intra-arterial delivery, the transient expression of integrins should be sufficient for diapedesis to occur. Since the capacity of MSCs to undergo functional transfection using pDNA is very low, we investigated an mRNA transfection method for the expression of transgenes in MSCs in order to overcome the limitations of the pDNA approach. Methods: Human mesenchymal stem cells (hMSC, PT-2501, Lonza) were thawed and cultured in medium MSCBM (PT-3238, Lonza) supplemented with 10% MCGS (PT-4106E, Lonza), L-glutamine (PT4107E, Lonza), and gentamicin sulfate (GA-1000, PT-4504E, Lonza). Cells were maintained in a humidified atmosphere at 37°C and 5% CO2 using 75 cm2 flasks. For transfection experiments, hMSCs were transferred to 24-well plates and seeded at a density of 15 000 cells/well. For transgene induction experiments, pDNA-eGFP (BD Biosciences) at a dose of 0.5 and 1.0 µg/well, and mRNA-eGFP (StemGent) at doses of 0.12, 0.25, and 0.5 µg/well were used. The Lipofectamine® 2000 (Invitrogen), TransIT-2020 (Mirus), and StemfectTM RNA Transfection Kit (StemGent) were used as transfection agents. After transfection, cells were maintained in culture conditions up to 21 days. Transfection efficiency was assessed by confocal microscopy using GFP fluorescent signal detection. Results: MSC pDNA-eGFP transfection results in a dramatically low efficiency, less than 1% of the cell population in each of the tested conditions. In contrast, mRNA-eGFP transfection resulted in an efficiency exceeding 95% in each of the tested conditions. This difference was highly statistically significant (P<0.001). Furthermore, cellular GFP level, and the persistence of transfection was dependent on the mRNA dose and the type of transfection agent. It was found that the dose of mRNA-eGFP 0.5 µg/well and the use of Lipofectamine was the most effective method with transgene expression up to three weeks. Conclusions: The mRNA transfection is a robust, clinically applicable tool for inducing the transient expression of transgenes in hMSCs, which are otherwise difficult to transfect by vectors that do not incorporate into the host genome. Using this method, application of engineered MSC could revolutionize regenerative medicine. Supported by a National Centre for Research and Development grant No 101 in ERA-NET NEURON project: “MEMS-IRBI”

Słowa kluczowe

EN

mRNA transfection; transient transgene; gene expression; man; mesenchymal stem cell; stem cell; functional property; transplantation; cerebral ischaemia; spinal cord injury; therapeutic effect; regenerative medicine

• Wydawca: -
• Czasopismo: Acta Neurobiologiae Experimentalis
• Rocznik: 2013
• Tom: 73
• Numer: 1
• Opis fizyczny: p.186

Twórcy

autor

Nowakowski A.

• NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

autor

Andrzejewska A.

• NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

autor

Kozlowska H.

• Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

autor

Walczak P.

• Division of MR Research, Russell H.Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, USA

autor

Lukomska B.

• NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

autor

Janowski M.

• NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland