Figure 5.

Silencing of GFP mediated by LNP-siRNA in well-defined primary MSCs in vitro is specific and dose dependent

Enrichment of PDGFRa/Sca1 MSCs population of GFP-expressing mice.

(A) Representative flow cytometric profiles of compact bone-derived mononuclear cells (top left FSC/SSC) and gated nucleated live cells (Hoechst positive/propidium iodide negative, top right) stained with CD45, TER119, CD31, biotinylated CD140 (PDGFRα, APA5, bottom left), SA-PE, and Sca-1 antibodies (bottom right). (B) Phase-contrast micrographs showing the morphology of a colony of MSC-derived cells sorted from 5,000 PDGFRα+/Sca-1+ cells at 7, 14, and 21 days after plating and culturing in MesenCult medium. Adipogenesis (bottom) as indicated by lipid vacuoles present intracellularly. (C) Osteogenesis of MSCs cultured for 14 days in DMEM or OM as detected by ALP (top) or von Kossa staining (bottom). (D–F) Data depict the presence of Tye 563-labeled siRNA (red) intracellularly in bone MSCs visualized using a Leica confocal microscope. Bone MSCs derived from GFP transgenic mice were treated with LNP-siGFP or LNP-siCtrl (PEG-DSG) at indicated doses. Bar graphs depict relative GFP expression of MSCs determined by flow cytometry at 4 (E) and 7 (F) days after treatment, following detection of MSCs using the appropriate antibodies. Data from one representative experiment of two independent experiments are shown (∗p < 0.05).