Figure 4.

Let-7 Upregulation Alone Is Insufficient to Drive hESC-β Cell Maturation

(A) Generation of iLET-7 cell line. Pre-let-7a/f (m) and pre-let-7b (m) are genomic sequences for let-7a/f and let-7b precursors with LIN28 binding region mutated (see Experimental Procedures). OE, overexpression.

(B) Schematic outlining let-7 induction protocol. Doxycycline treatment was from d14 to d27. Fluorescence-activated cell sorting was performed on d20. GSIS and qRT-PCR were performed on d27.

(C) qRT-PCR analysis of representative let-7 family members in iLET-7 hESC eBCs. Doxycycline treatment as in (B). n = 6 independent samples for Dox–, n = 5 independent samples for Dox+. Values are average ± SEM. Statistical significance was calculated using unpaired two-tailed t test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; n.s., not significant.

(D) qRT-PCR analysis of LIN28B and HMGA2 expression in iLET-7 hESC eBCs. Doxycycline treatment, number of replicates and statistics as in (C).

(E) Static GSIS of iLET-7. Doxycycline treatment as in (B). n = 11 independent samples for Dox–, n = 10 independent samples for Dox+. Values are average ± SEM. Statistical significance was calculated using unpaired two-tailed t test. n.s., not significant. Compare with iCrLIN28B in Figure S4E.

(F) qRT-PCR analysis of selected gene expression in iLET-7 hESC eBCs. Dox treatment, number of replicates, and statistics as in (C).

See also Figure S4.