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. Author manuscript; available in PMC: 2020 Oct 3.
Published in final edited form as: Cell Stem Cell. 2019 Sep 12;25(4):486–500.e9. doi: 10.1016/j.stem.2019.08.005

Figure 3. Sustained Transgene Expression Differentiates Complete from Partial Reprogramming.

Figure 3.

(A) Hb9::GFP+ cells with fibroblast (top) or neuronal (bottom) morphology at 17 dpi. Scale bars represent 20 μm.

(B) Percentage of Hb9::GFP+ cells of all viable cells measured by flow cytometry at 8 dpi. n = 6 independent conversions per condition. Mean ± SEM; one-way ANOVA.

(C) Percentage of Hb9::GFP+ cells with neuronal morphology of total Hb9::GFP+ cells at 17 dpi. n = 9 independent conversions per condition. Mean ± SEM; one-way ANOVA.

(D) Relative gene expression of cells collected at 14 dpi sorted based on No, Low, or Bright Hb9::GFP expression. Bright Hb9::GFP, cells in the top 50% of Hb9::GFP in the 6F condition. Gene expression was calculated based on qRT-PCR data. The expression level that was highest among the three conditions was set to 1 and used to normalize levels for the other two conditions. n = 2 independent experiments for each condition.

(E) Relative expression for single cells with either fibroblast (n = 16) or neuronal (n = 39) morphology for qPCR assays for endogenous Ngn2 and Isl1 and viral Isl1 (vIsl1). Median ± interquartile range is shown; Mann-Whitney test.

(F) Relative Isl1-GFP intensity in all viable cells (all) and HyperP infected with Isl1-GFP and 6F or 6FDDRR measured by flow cytometry at 4 dpi. n = 4–6 independent transductions per condition. Mean ± SEM; one-way ANOVA.

(G) Percentage of Isl1-GFP+ cells in all viable cells (all) and HyperP measured by flow cytometry at 4 dpi. Isl1-GFP+ determined by expression exceeding fluorescein isothiocyanate (FITC) values for untransfected cells is shown. n = 6 independent transductions per condition. Mean ± SEM; one-way ANOVA.

(H) Relative integrations of Isl1-GFP and NIL viruses in cells collected at 4 dpi. Relative integrations determined by qPCR are shown. Delta Ct of transgene calculated by difference of Ct between transgene and endogenous genomic region is shown. Relative integrations calculated by normalizing to NIL condition are shown. n = 3 independent transductions per condition. Mean ± SEM; one-way ANOVA.

Significance summary: p > 0.05 (ns); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; and ****p ≤ 0.0001.