Figure 2.

A micro-manipulated single PCFU is sufficient to give rise to a colony expressing the three major pancreas lineages, and identification of SOX9⁺/PDX1⁺/NKX6.1⁺ cells in endogenous ducts

(A) Experimental diagram.

(B) Time course bright-field imaging of a single PCFU grown into a colony. Scale bar, 20 μm (on day 0) and 50 μm (for all other days).

(C and D) (C) % PCFUs and (D) mean diameter of colonies grown from 1 cell per well vs. multiple cells per well; mean ± SEM from 4 independent experiments using 3 donor tissues. Paired t test determined significance.

(E) Microfluidic qRT-PCR analysis of colonies grown from unsorted cells plated at multiple cells per well (black) (n = 24 colonies, 3 independent experiments from 2 donors), vs. 1 cell per well (red) (n = 22 colonies); mean ± SD. Significance was determined by two-way ANOVA with Sidak’s multiple comparison.

(F) IF staining of Pan-CK (green), CK19 (red), and MUC1 (white) in human pancreas. The image contains interlobular, intralobular, and intercalated ducts. Scale bar, 50 μm.

(G) The same region in a sequential slide to (F) is stained with NKX6.1 (green), PDX1 (white), and SOX9 (red). Insets 1 and 2 highlight an interlobular and an intercalated duct, respectively; both contained TP cells (yellow arrows point to representatives of SOX9⁺PDX1⁺NKX6.1⁺ cells) and non-TP cells (red arrow points to representative of a SOX9⁺PDX1⁺NKX6.1⁻ cell). Scale bar, 50 μm (insets enlarged 4×).

(H) Quantification of % TP cells among total cells within stitched images; mean ± SEM (10.2% ± 2.6%), N = 4 donor tissues. See also Figures S1F and S2.