Combined expression of pTFs promotes transdifferentiation efficiency, however only their sequential expression increases β-cell maturation.

<p>Cultured adult human liver cells were infected with <i>Ad-CMV-Pdx1</i> (1000 MOI), <i>Ad-CMV-Pax-4</i> (100 MOI) and <i>Ad-CMV-Mafa</i> (10 MOI) together or in a sequential manner as summarized in (A) (treatments B–E) or with control virus (<i>Ad-CMV-β-gal</i>, 1000 MOI, treatment A), and analyzed for their pancreatic differentiation six days later. (B) Immunofluorescence staining of treated human liver cells for insulin (red). Nuclei were stained with DAPI (blue), original magnification X20. The percent of insulin positive cells were calculated by counting at least 1000 positive cells from at least two independent experiments preformed in cells isolated from different donors. Insulin (and or pro-insulin) (C) and c-peptide (D) secretion were measured by static incubation of the cells for 15 min at 2 and 17.5 mM glucose in KRB. *<i>p<</i>0.05, **<i>p<</i>0.01, compared to control virus treated cells; n≥12 in 5 independent experiments preformed in cells isolated from different donors. (E) Quantitative Real-Time PCR analysis for gene expression of endogenous pTFs. CT values are normalized to β-actin gene expression within the same cDNA sample. Results are presented as relative levels of the mean±SE of the relative expression versus control virus treated cells. *<i>p<0.05</i>, n≥8 in 4 independent experiments preformed in cells isolated from different donors. The arrow points to the specific decrease in Isl1 expression level under the C-protocol, sequential and direct hierarchical administration of pTFs.