Fig 1.
In vitro expanded primary epithelial cells from human fetal small and large intestine express stem cell markers.
A-F, top panels: small intestine, bottom panels: large intestine (A) Hematoxylin and eosin paraffin sections of normal fetal intestine at 23 weeks of gestation. (B) Intestinal cells expanded on the feeder layer (yellow outline). (C-F) Representative analyses of primary and expanded cells at passages 4, 5 or 6 with the percentage of gated live cells provided. (C) Flow cytometric analysis (dot plots) of freshly isolated primary intestinal cells. Gating strategy identifies total live cells (left panels) and EPCAM+ cells, excluding blood cells (CD45+CD235a+) (right panels). (D) Flow cytometric analysis of expanded cells. Gating strategy identifies total live cells (left panels) and EPCAM+ cells, excluding rodent feeder layer cells (HLA−) (right panels). (E) Flow cytometric analysis (5% probability contour plots) of primary and expanded intestinal cells, stained with EPCAM and CD133.1. (F) Expanded intestinal cells also express other putative stem cell markers. Histograms represent unstained control (blue) and stained with CD166 or CD24 (red) of live EPCAM+ cells. Scale bars = 100μm for panel A and B.
Fig 2.
Expanded small and large intestinal cells show different cell surface marker expression.
(A) Flow cytometric analysis of CD133.1, CD13 and CD66c expression on expanded cells from one paired SI and LI sample (SI and LI depicted here originated from the same tissue source). The histograms show one representative set of three paired samples examined presenting unstained control (blue) and stained live, EPCAM+ cells (red). For CD66c, the percentage of low and high expressing cells is provided. All markers were PE-labeled. The same control was used for SI CD13 and CD66c as well as LI CD133 and CD13 because the staining was done at the same time. (B) Corresponding staining index of three paired samples showing reproducible results between the paired SI and LI samples (1, 2 and 3) (staining index = D/W, where D is the distance between the positive and negative populations and W is two standard deviations of the negative population).
Table 1.
Limiting Dilution Analysis (LDA) indicates that opposing CD66c expression separates colony-forming ability in epithelial cells expanded from SI and LI.
Fig 3.
Gene array analysis revealed differing molecular signatures between cells expanded from SI and LI.
(A) Heat map of differentially expressed transcripts of the expanded epithelial cells from SI versus LI. Unsupervised clustering was performed on log2 gene expression values (K-means algorithm) with q<0.05 (FDR = 5%) cutoff. Expression values are scaled for minimum (blue) and maximum (red) intensity values. Expanded cells formed distinct clusters based on cell origin (SI or LI). (B-D) Histograms depict significant (q<0.05) differential expression of individual genes between cells expanded from SI and LI classified into canonical pathways using Ingenuity Pathways Analysis (see Methods). All values are reported as mean (box) plus standard deviation (error bar) of raw intensity for each transcript. List of gene names can be found in S2 Table.
Fig 4.
Three-dimensional differentiation assay for cells expanded from SI and LI.
(A) Experimental design of three-dimensional assay. Expanded intestinal cells were embedded as single cells in Matrigel, first expanded in Stem Cell (SC) media, then treated further with SC media, Differentiation media or Differentiation media + DAPT (GSI). (B) In vitro and H&E pictures of SI (top) and LI (bottom) organoids in each of the three treatments. One paired set of SI and LI organoids is representative of three sets examined. Scale bar = 50μm.
Fig 5.
Immunofluorescent staining of intestinal organoids confirms multipotentiality and differentiation potential.
(A) SI and (B) LI organoids from one paired sample stained for epithelial marker, EPCAM (green), goblet cell marker, MUC2 (red) and enteroendocrine marker, CHGA (red) treated with SC media, Differentiation media (Diff) or Diff media + DAPT. Counterstain, Hoechst 33342. White arrows indicate positive cells. Scale bar = 50μm. (C) Quantification of percent goblet or enteroendocrine cells of total cells counted per field of view. Not enough enteroendocrine cells were present in the organoids derived from LI for quantification. This paired set serves as a representative of two sets examined.
Fig 6.
Small and large intestine organoids differ in response to differentiation cues.
Real-time PCR analysis of (A&C) SI and (B&D) LI organoids derived from expanded cells and treated with SC media, Differentiation (Diff) or Diff media + DAPT. SC media treatment values used as control and set to 1. Diff media and Diff media + DAPT expression values are relative to SC media. See media key in panel A. (E) Select marker expression comparison between SI and LI organoids in the three treatments. SI values used as control and set to 1. LI organoid expression values are relative to SI organoids. See text for full gene names. Expression was normalized to GAPDH mRNA. Error bars represent upper and lower error limits based on replicate variability (*P<0.05, **P<0.01, ***P<0.001, # no expression detected). (n = 3 wells per sample/primer pair). One paired SI and LI set serves as a representative of all paired sets examined.