Figure 1.
Derivation of cPP Cell Lines from hESC and hiPSC
(A) Pancreatic progenitors generated after 15 days of differentiation using the STEMdiff directed differentiation kit (PPd15 cells) were plated and expanded on a layer of 3T3-J2 feeder cells in medium supplemented with the indicated growth factors and signaling inhibitors.
(B) Intracellular flow cytometric analysis for PDX1 and NKX6-1 at days 8, 10, and 15 of differentiation using H9 hESCs.
(C) Phase-contrast images of cPP cells passaged as aggregates (left) and as single cells (right). Scale bar, 100 μm.
(D) Gene expression measured by qRT-PCR using samples harvested from PPd15 cells and cPP cells at early (6–8), middle (11–13), and late (14–18) passages. Cells were derived from both AK6-13 hiPSC and H9 hESC. Gene expression in definitive endoderm (H9 hESCs after 4 days STEMdiff differentiation) is shown for comparison. Values are plotted on a log2 scale and error bars represent the SE of three technical replicates. ND, not detected.
(E) Immunofluorescence staining of cPP cells for key pancreatic transcription factors. Scale bar, 100 μm.
(F) Intracellular flow cytometric analysis of cPP cells for PDX1. Gray dots represent control cells stained with isotype control antibodies.