Figure 5

The CsH-mediated increase in the efficiency of LV transduction of primitive murine HSPCs persists through transplantation. LSK (Lin⁻cKit⁺Sca1⁺) cells were isolated from the BM of CD45.2 mice, transduced with a mix of LVs designed to express ZsGreen, mCherry or RFP670 fluorescent reporters using spinoculation (see Materials and methods) (MOI values: ZsGreen – 42, mCherry – 28, and RFP670–17), incubated in the presence of CsH/DMSO for 24 hours and injected into lethally irradiated CD45.1 recipients together with whole BM CD45.1 competitor cells (3 mice in each group). The expression of fluorescent reporters and chimerism in the peripheral blood (PB) were assessed at various time points using flow cytometry. (A) Representative plots showing the proportion of donor-derived cells and the expression of fluorescent reporters in a mouse from the control group (top panel) vs the CsH group (bottom panel) 16 weeks post-injection. Cells expressing all three fluorescent reporters were identified by assessing the proportion of cells positive for mCherry out of the population of cells positive for both ZsGreen and RFP670. (B) The proportion of cells expressing mCherry out of total donor-derived (CD45.2⁺) cells. Data were collected from three independent experiments (n = 3), and triplicate technical replicates were used within the experiments. (C–G) Quantification of the percentages of CD45.2⁺ cells (C), cells expressing each fluorescent protein (D–F), and cells expressing all three reporters (G) in the PB of control or CsH mice at various time points post-transplantation. The results are presented as the means ± SDs. Statistical significance was calculated by an unpaired t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.