Fig. 6

Transfection of stem cells with mRNA nanoparticles can promote their expansion and self-renewal. a Targeting of CD105 enables specific transfection of HSC CD34⁺ cells. Cells were left untreated, or transfected with eGFP-encoding mRNA in nanoparticles (NPs) coated with PGA coupled to a control antibody or anti-CD105. Transfection efficiency was assayed by flow cytometry after 24 h. b NP transfection efficiency in CD34⁺ samples from three independent donors. Viability is shown in c. d Expansion of CD34 + PBSCs after NP-transfection. e Phenotypical characterization of PBSC-derived CD34⁺ subpopulations after 2 days in culture. Cells were either transfected with eGFP NPs on day 1 or left unmodified. Gating is indicated in brackets on top of each column. f Summary bar graph showing mean frequencies and SE of primitive Hematopoietic Stem Cells (HSCs), Multipotent Progenitors (MPPs), Lymphoid-primed Multipotent Progenitors (LMPs), and Early Myeloid Progenitors (EMPs). PBSCs from four independent donors were analyzed. Error bars represent mean ± SE. g Colony output of sort-purified GFP-NP transfected versus unmodified CD34⁺ cells from day 7 cultures (n = 3 cultures from independent donors); n.s., non-significant. Arising colonies were identified as colony forming unit (CFU) granulocyte (CFU-G), macrophage (CFU-M), granulocyte-macrophage (CFU-GM) and burst forming unit-erythrocyte (BFU-E). Colonies consisting of erythroid and myeloid cells were scored as CFU-MIX; n.s., non-significant. Error bars represent mean ± SE. h Representative images of CFU-MIX colonies from untransfected and GFP-NP-transfected CD34⁺ cells (×4-magnification; scale bar 1000 µm). i qPCR measurements of NP-delivered Musashi-2 (MSI2) mRNA expression over time. Error bars represent mean ± SE. j Comparison of CD133 and CD34 expression in HSCs transfected with control GFP mRNA-NPs versus MSI2 mRNA-NPs, assessed by flow cytometry 8 days after NP exposure and cell expansion. Data represent two independent experiments conducted in triplicate. k Cellular fold expansion of CD34− (differentiated) and CD34⁺ CD133⁺ (progenitor) cells. Bar graphs show mean and SE of three independent experiments. Data represent two independent experiments conducted in triplicate. l Colony forming unit outputs of untransfected versus MSI2-NP-transfected HSCs (n = 3 cultures from independent donors); Pairwise differences between groups were analyzed with the unpaired, two-tailed Student’s t Test. *P = 0.049, **P = 0.012, ***P = 0.011; n.s., non-significant. Error bars represent mean ± SE. m Representative images of colonies from untransfected and MSI2-NP-transfected CD34⁺ cells (scale bar, 300 µm)