1. Volume 138, Issue 3, Pages 1012-1021.e5 (March 2010)
Immortalized Epithelial Cells Derived From Human Colon Biopsies Express Stem Cell Markers and Differentiate In Vitro 
Andres I. Roig, Ugur Eskiocak, Suzie K. Hight, Sang Bum Kim, Oliver Delgado, Rhonda F. Souza, Stuart J. Spechler, Woodring E. Wright, Jerry W. Shay 
Gastroenterology 
Volume 138, Issue 3, Pages e5 (March 2010)
DOI: /j.gastro
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2. Supplementary Figure 1
Western blot demonstrating mucin 1 (MUC1) expression in human colonic epithelial cell immortalized with cyclin-dependent kinase-4 and catalytic component of human telomerase. DLD-1 colon cancer epithelial cell lines used as a positive control and BJ skin fibroblast as negative control (data not shown).
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3. Supplementary Figure 2
Percent expression of colonic epithelial cell markers obtained by quantification of immunofluorescent markers in human colonic epithelial cells immortalized with cyclin-dependent kinase-4 and catalytic component of human telomerase from both patients. Chromogranin A (CGA) quantification obtained in log-growth cells while quantification of other markers obtained in growth arrested cells.
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4. Supplementary Figure 3
Western blots demonstrating cytoplasmic and nuclear β-catenin levels in log-growth and growth-arrested (BIO) cells.
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5. Supplementary Figure 4
Microarray expression data of a subset of stem cell markers in human colonic epithelial cell immortalized with cyclin-dependent kinase-4 and catalytic component of human telomerase in log-growth.
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6. Supplementary Figure 5
Western blots demonstrating stem cell marker expression in human colonic epithelial cell immortalized with cyclin-dependent kinase-4 and catalytic component of human telomerase. (A) CD29 (B) CD44.
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7. Supplementary Figure 6
Transmission electron microscope image of log-growth cells illustrating paucity of intracellular structures, brush border, or microvilli, supporting an undifferentiated nature of cells.
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8. Supplementary Figure 7
Transmission electron microscope image demonstrating ultrastructural junctional elements of immortalized human colonic epithelial cell line 1 in the growth arrested state (BIO treated). Left panel illustrates presence of organelle structure that include mitochondria, endoplasmic reticulum, and Golgi (arrow); right panel suggests presence of immature tripartite junctions.
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9. Supplementary Figure 8
Human colonic epithelial cell immortalized with cyclin-dependent kinase-4 and catalytic component of human telomerase (HCEC CTs) are karyotypically diploid and do not have tumorigenic features. (A) Karyotype of HCEC 1CT. (B) HCEC CTs do not make large colonies in soft agar (top panel) as do the transformed colonic epithelial cell line HCT 116.
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10. Figure 1
Crypt extraction sequence. Crypts are extracted from processed biopsies and seeded onto a Primaria dish with colonic fibroblast feeder layers. The dishes are immediately placed in 2% to 5% oxygen tension. Ten days after seeding, nests of epithelial cells can be identified. Scale bars = 50 microns for 10 day explant; 75 microns for liberated crypt; 150 microns for crypts panel.
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11. Figure 2
Immortalization of colonic epithelial cells. (A, B) Growth curves for cells derived from patient 1 and patient 2. Normal unimmortalized cells undergo replicative senescence (C). Expression of Cdk4 and hTERT maintains a compact and healthy appearance of HCEC CTs even after 90 PD (D) compared to non-infected HCEC CTs at PD 40 (C). Scale bars = 50 microns for (C) and (D).
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12. Figure 3
Cdk4 and telomerase expression in immortalized HCEC CTs. (A) Western blots showing prominent Cdk4 and p16 bands. (B) Telomere restriction fragment length analysis gel showing overall increased telomere length in immortalized HCECs compared to normal HCECs that progressively show telomere length decreases until senescence. (C) Telomeric repeat amplification protocol assay showing positive telomerase activity in both HCEC 1CT and HCEC 2CT cell lines (2500 cell equivalents) and lack of telomerase activity in the parental HCEC1 and HCEC2 cell strains compared to a dilution series of HeLa cells. ITAS, internal telomerase assay standard.
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13. Figure 4
Representative images characterizing HCEC CTs in log-growth and growth arresting conditions (BIO treated). (A) Appearance of HCEC CTs in log-growth. (B, C) Positive expression of proliferating cell nuclear antigen (PCNA) status (green) and α-smooth muscle actin (α-SMA) (red), respectively, in log-growth cells. (D) Appearance of HCEC CTs in growth arrested state; arrows indicate circular openings of self-organizing structures. (E, F) No detectable expression of PCNA and α-SMA, respectively, in growth arrested cells. Blue nuclei, DAPI; Scale bars = 50 microns for panels (A) and (D); 10 microns for (B, C); (E, F).
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14. Figure 5
Expression of epithelial markers in growth arrested (BIO treated) cells (cytokeratin 18, cytokeratin 20, zonula occludens-1 [ZO-1], β-catenin, A33, villin, mucin [MUC] 1, MUC2, and dipeptyl peptidase 4; [A−I]), and in log-growth cells (Chromogranin A; J). Rhodamine and fluorescein isothiocyanate (FITC) controls (K, L). Scale bars = 10 μm for all images.
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15. Figure 6
Stem cell markers in log-growth HCEC CTs. (A, B) Cytoplasmic (green) LGR5 staining in HCEC 1CT and HCEC 2CT, respectively. (C) Negative staining for LGR5 in C26Ci human colon fibroblasts. (D, E) Nuclear (red) BMI1 staining in HCEC 1CT and HCEC 2CT, respectively; (F) Greatly reduced BMI1 in control colonic fibroblasts. Blue nuclei, DAPI; scale bars = 10 microns for all images.
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16. Figure 7
Multicellular (cyst-like) structure formation and differentiation in three-dimensional Matrigel culture. (A) In Matrigel culture, individual HCEC CTs proliferate and form progressively enlarging multicellular structures (20× magnification; Evos microscope). Small self-organizing multicellular structures (day 11) show evidence of (mucin [MUC]2 (green), zonula occludens-1 (ZO-1) (red), and general nuclear staining (blue) (B, C) (C). Larger cyst-like structures (day 18) exhibit villin expression (red) lining the central luminal portions (D), and MUC2 (red) in a subset of cells comprising the cysts (E). Scale bars = 20 microns for fluorescent images.
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