FIG. 1.
The serum-free, conditioned medium-free, 7F culture system allows formation of colonies (organoids) composed of the three major pancreatic lineages. (A) Colony number and (B) colony-forming efficiency are shown for the standard (n = 11) and 7F (n = 27) culture systems after 3 weeks in culture. n.s., not significant (P > 0.05) determined by t-test. Data represent mean ± SEM. (C) Representative photomicrograph of 3-week-old cystic colonies grown in the standard or 7F culture system. Bar: 100 μm. (D) Single-colony microfluidic qRT-PCR analysis for ductal, progenitor, acinar, and endocrine markers. Each column is from a single colony. cDNA from adult pancreas (Ad. Panc.) and day-6 embryoid bodies from a mouse embryonic stem cell line (EB d6) [29] were used as positive controls. H2O was used as negative control. Shown is one representative experiment of three with the same trend. (E) Representative photomicrographs of whole-mount immunofluorescent staining for ductal (Mucin1, Sox9), acinar (Amylase), endocrine (Ghrelin), and pancreatic progenitor (Pdx1) markers in colonies grown in 7F. Bar: 50 μm. (F) Representative TEM images showing ductal cell characteristics such as a multi-lobed nucleus (white asterisk), microvilli (black arrow) facing the lumen and desmosomes (black arrowhead) at cell–cell junctions. The dotted box is shown enlarged on top. Bars: 1 and 0.5 μm. 7F, seven factor; cDNA, complementary DNA; qRT-PCR, quantitative reverse transcription–polymerase chain reaction; SEM, standard error of the mean; TEM, transmission electron microscopy.