Figure 4.

A Layer of 3T3-Feeder Cells and Exogenous Signaling Molecules Are Required for the Maintenance and Expansion of cPP Cells

(A) Phase-contrast images of H9 and AK6-13 cPP cells after 7 days culture in complete medium on 3T3-feeder cells plated at densities of 5 × 10⁴, 2.5 × 10⁴, and 1.25 × 10⁴ cells/cm². Scale bar, 100 μm.

(B) Gene expression measured by qRT-PCR for samples harvested from cultures in (A) for endocrine (NGN3 and NKX2-2), ductal (KRT19 and CA2), and acinar (CPA1 and AMY2B) marker genes. Error bars represent the SE of three technical replicates.

(C) Phase-contrast images of cPP cells cultured for 6 days in complete medium with individual components omitted. Scale bar, 100 μm.

(D) PDX1 and SOX9 expression measured by qRT-PCR for samples harvested in (C). Error bars represent the SE of three technical replicates.

(E) Microbioreactor array (MBA) screening of factors required to propagate PDX1+SOX9+ cPP cells. Effects of reducing or removing selected factors (EGF, RA, DAPT) from complete medium containing all factors at the following levels: EGF (50 ng/mL), RA (3 μM), DAPT (1 μM), SB431542 (10 μM), and FGF10 (50 ng/mL). Top panels: effects on total nuclei per chamber, and PDX1 and SOX9 mean nuclear intensity. Lower panels: effects on the total number of PDX1+SOX9+ cells per chamber and percentage of PDX1+SOX9+ cells. Data represent the mean of ten chambers within a column treated with the given condition ± the SE.

(F) Heatmap showing RNA-seq expression levels of components of signaling pathways that regulate cPP proliferation: EGF (EGFR), FGF10 (FGFR1-4, 6 and FGFRL2), RA (RARA, RARB, RARG, RXRA, RXRB, and RXRG), SB431542 (ACVR1B [ALK4], TGFBR1 [ALK5], and ACVR1C [ALK7]), and DAPT (NOTCH1-4 and its ligands DLL1,3,4 and JAG1,2). Levels are shown relative to those observed across all 25 tissues shown in Figure 3A.