Successfully Transfected Primary Peripherally Mobilized Human CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs) Demonstrate Increased Susceptibility to Retroviral Infection 

Authors:
Jeffrey Sebrow , Stephen P Goff, Daniel O Griffin             
In:
Source: Virol J
Publication Date: (2020)
Issue: 17: 1
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
Jurkat
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
293T
Species: human
Tissue Origin: kidney
Culture Media:
Platform:
4D-Nucleofector™ X-Unit
Experiment

Peripherally mobilized CD34+ HSPCs from individual donors from AllCells/ Lonza were  cultured in serum free media (X-VIVO 20), with stem cell factor (SCF), Flt3 ligand (FLT3L).
thrombopoietin (TPO) at 100 ng/ml.. CD34+ cells were also transfected in cuvettes. 50,000 cells were suspended in 20 ul of transfection reagent, and 2 µg of pmCherry-C1 DNA were added. The cells were subjected to electroporation using the Lonza 4D-Nucleofector X Unit with the Lonza P3 Primary Cell 4D-Nucleofector X Kit S according to the Lonza Amaxa 4D-Nucleofector Protocol for unstimulated CD34+ cells (https://bioscience. lonza.com). After the transfection process, 180 ul of culture media was added to each cuvette well and mixed with the transfection solution. A total of 90 ul of media/solution was removed and placed in culture wells containing 160 ul of media.

Abstract

Transfection, the process of introducing purified nucleic acids into cells, and viral transduction, viral-mediated nucleic acid transfer, are two commonly utilized techniques for gene delivery in the research setting. Transfection allows purified nucleic acid to be introduced into target cells through chemical-based techniques, nonchemical methods or particle-based methods, while viral transduction employs genomes or vectors based on adenoviruses, retroviruses (e.g. lentiviruses), adeno-associated viruses, or hybrid viruses. Transfected DNAs are often tested for potential effects on subsequent transduction, but it is not clear whether transfection itself rather than the particular nucleic acid being introduced might impact subsequent viral transfection. We observed a significant association between successfully transfected mobilized peripheral blood CD34+ human stem and progenitor cells (HSPCs) and permissiveness to subsequent lentiviral transduction, which was not evident in other cells such as 293 T cells and Jurkat cells. This association, apparently specific to CD34+ human stem and progenitor cells (HSPCs), is critical to both research and clinical applications as these cells are a frequent target of transfection and viral transduction owing to the durable nature of these cells in living systems. This finding may also present a significant opportunity to enhance the success of viral transduction for clinical applications.