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Immortalized Epithelial Cells Derived From Human Colon Biopsies Express Stem Cell Markers and Differentiate In Vitro
GASTROENTEROLOGY 2010;138:1012–1021
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,* and JERRY W. SHAY* *Department of Cell Biology; ‡Department of Medicine, Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas; and § Veterans Affairs North Texas Health Care System, Dallas, Texas
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BACKGROUND & AIMS: Long-term propagation of human colonic epithelial cells (HCEC) of adult origin has been a challenge; currently used HCEC lines are of malignant origin and/or contain multiple cytogenetic changes. We sought to immortalize human colon biopsyderived cells expressing stem cell markers and retaining multilineage epithelial differentiation capability. METHODS: We isolated and cultured cells from biopsy samples of 2 patients undergoing routine screening colonoscopy. Cells were immortalized by expression of the nononcogenic proteins cyclin-dependent kinase 4 (Cdk4) and the catalytic component of human telomerase (hTERT) and maintained for more than 1 year in culture. RESULTS: The actively proliferating HCECs expressed the mesenchymal markers vimentin and ␣-smooth muscle actin. Upon growth arrest, cells assumed a cuboidal shape, decreased their mesenchymal features, and expressed markers of colonic epithelial cells such as cytokeratin 18, zonula occludens-1, mucins-1 and -2, antigen A33, and dipeptidyl peptidase 4. Immortalized cells expressed stem cell markers that included LGR5, BMI1, CD29, and CD44. When placed in Matrigel in the absence of a mesenchymal feeder layer, individual cells divided and formed self-organizing, cyst-like structures; a subset of cells exhibited mucin-2 or polarized villin staining. CONCLUSIONS: We established immortalized HCECs that are capable of self-renewal and multilineage differentiation. These cells should serve as valuable reagents for studying colon stem cell biology, differentiation, and pathogenesis. Keywords: Stem/Progenitor Cells; Epithelial Mesenchymal Transition; Self-Renewal; Multilineage Differentiation.
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he long-term propagation of human colonic epithelial cells (HCEC) of adult origin has historically been challenging. Although progress has taken place in the development of methods to culture HCECs, including the description of some long-term culture models, these techniques and cell lines have not gained wide acceptance in the scientific community possibly as a result of inherent technical difficulties in extracting and maintaining viable cells.1–10 A majority of the currently used HCEC lines are of malignant origin and/or contain multiple
cytogenetic changes. Although these cell lines have been highly informative in many aspects of intestinal cell biology research, established adult HCEC lines not derived from tumor specimens and that are karyotypically normal are needed to conduct research that would be more difficult to perform with the currently established cell lines. For example, cell culture models of nontransformed adult HCECs could be used to test current paradigms of colorectal cancer progression. Creating de novo genetic changes, via a candidate or screening approach, would permit validation of known or novel mutations that confer a growth advantage to cells. This could then serve as a basis for the development and application of agents to test on cells, to determine if they have an effect on the aberrant pathways leading to transformation, and potentially permit the discovery of new drugs that mitigate colorectal cancer development. In the present studies, we demonstrate the long-term growth of adult human colonic epithelial progenitor cells extracted from colonic biopsy specimens taken from patients undergoing screening colonoscopy. We show that in low-oxygen culture conditions, cells replicate and can be immortalized with the nononcogenic proteins cyclindependent kinase 4 ([Cdk4], bypasses cell culture associated stresses that frequently lead to premature senescence) and the catalytic component of the human ribonucleoprotein enzyme telomerase ([hTERT]; maintains telomere length and prevents replicative senescence). Low oxygen tension (2%⫺5% oxygen) has previously been shown to extend the replicative lifespan of many human cell types (fibroblasts and epithelial cells) by delaying the stress-induced senescence that results from normal atmospheric (21%) oxygen tension.11–14 Cdk4 and hTERT have previously been used to immortalize various epithelial cell types without conferring tuAbbreviations used in this paper: BIO, bromoindirubin-3-oxime; 2D, two-dimensional; 3D, three-dimensional; Cdk4, cyclin-dependent kinase-4; EGF, epithelial growth factor; GSK-3, glycogen synthase kinase-3; HCEC CT, human colonic epithelial cell immortalized with Cdk4 and hTERT; hTERT, catalytic component of human telomerase; MUC, mucin; PD, population doublings; ZO-1, zonula occludens-1. © 2010 by the AGA Institute 0016-5085/10/$36.00 doi:10.1053/j.gastro.2009.11.052
morigenic properties.15–18 Characterization of the immortalized colonic cells reveals that they exhibit morphological features and markers characteristic of epithelial cells, particularly when growth is arrested as a result from serum deprivation, reduced epithelial growth factor (EGF), and treatment with glycogen synthase kinase-3  (GSK-3) inhibitors. In addition, the cells display LGR5 and BMI1, proteins marking the stem cells of the mice gastrointestinal tract and that also have been identified in the cells of human colonic crypts,19 –23 as well as other proteins marking the rapidly proliferating human colonic epithelial cells in vivo (CD29 and CD44).24,25 Importantly, individual immortalized cells can proliferate and subsequently differentiate into multicellular structures and express multilineage colonic differentiation specific markers, similar to self-organizing structures formed by mouse intestinal epithelial cells in 3-dimensional (3D) culture.26,27
Methods Colonic Tissues This study was approved by the Institutional Review Board at the Dallas VA Medical Center. Colon biopsies (20⫺30 samples, ⬃0.5 cm3) from tissue not involved with endoscopically visible adenomas were obtained from patients undergoing routine screening colonoscopy after obtaining informed consent.
Growth Media and Tissue Culture Substrate Cells were routinely grown on basal X media (HyClone, Logan, UT) supplemented with EGF (25 ng/ mL) (PeproTech, Inc, Rocky Hill, NJ), hydrocortisone (1 g/mL), insulin (10 g/mL), transferrin (2 g/mL), sodium selenite (5 nanomolar) (all from Sigma, St Louis, MO), 2% cosmic calf serum (HyClone), and gentamicin sulfate (50 g/mL) (Gemini Bio-Products, West Sacramento, CA). Cells were cultured in Primaria flasks (BD Biosciences, San Jose, CA) and grown in 2%⫺5% oxygen and 7% carbon dioxide. HCT 116, BJ human skin fibroblasts, HeLa cells, and immortalized human colonic fibroblast cells (C26Ci, population doubling 150)28 were maintained in X media supplemented with 10% cosmic calf serum (HyClone). C26Ci cells were treated with 10 g/mL mitomycin C (Sigma) for 2 hours and used as feeder layers from the point of initial crypt attachment until the first passage.
Cell Isolation and Immortalization The intestinal cell isolation techniques previously described were used with some modifications.29,30 Colonic biopsies were immersed in cold X medium, brought to the laboratory within 40⫺60 minutes after colonoscopy, copiously washed with phosphate-buffered saline containing antibiotic/antimycotic solution (Gemini BioProducts), and cut into multiple small pieces ( ⬃1 mm in
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size). After enzymatic digestion with collagenase 150 U/mL (Worthington Biochemical, Lakewood, NJ) and dispase 40 g/mL (Roche, Germany), crypts were resuspended in X medium with growth supplements including 2% serum, and plated in Primaria culture dishes seeded 48 hours previously with 50% confluent colonic fibroblast feeder layers. During the first 10 days after attachment, cells were fed every 3 days, reducing the serum by 1% each change until 0% to prevent growth of unwanted cells such as fibroblasts. Once small nests of expanding epithelial cells were easily observed, cells were transduced with retroviral Cdk4 and hTERT as described previously.17 When numerous cuboidal-appearing cell nests were observed (3⫺4 weeks after initial crypt seeding), cells were reseeded on Primaria flasks. Feeder layers were not needed or used for routine tissue culture after the first passage.
Growth Arrest Conditions, Telomeric Repeat Amplification Protocol, Western Blotting, Immunocytochemistry, Tumorigenic Assays, Electron Microscopy, Matrigel Culture Please refer to Supplementary Materials and Methods section for these standard procedures.
Results Primary Culture and Immortalization of Cells Figure 1 describes the crypt extraction procedure. Enzymatic digestion yielded a higher number of viable crypts compared with chelation methods. Initial expansion of cells is limited in normoxic/atmospheric (21%) conditions and in conventional plastic culture dishes. Thus, optimized culture conditions include low oxygen tension (2%⫺5% O2),31 modified substrate (Primaria plastic culture dishes32), and initial growth of cells in half confluent human colon fibroblast-feeder layers. After the first passage, cells are subcultured in the presence of 2% serum, but in the absence of feeder layers. Cells from these primary cultures can continuously divide in these conditions for approximately 40 population doublings (PD) (Figure 2A and B). Cells obtained from the 2 patient specimens were transduced with both Cdk4 and hTERT. The resulting immortalized cells are termed human colonic epithelial cell (HCEC) 1CT (patient 1; “C” for Cdk4 and “T” for telomerase) and HCEC 2CT (patient 2). Both lines have been continuously subcultured for ⬎100 PD, are able to be cryopreserved, and have been reintroduced into cell cultures without complications (Figure 2A and B). Cessation of growth of nonimmortalized cells is accompanied by a flattened and enlarged morphological phenotype typical of cells undergoing senescence (Figure 2C, right panel). Compared to the flattened and enlarged shape of the nonimmortalized controls at senescence, the compact appearance of immortalized HCEC CTs PD 90 (Figure 2D, right panel) is similar to the morphology of
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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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earlier passaged cells in log-phase growth (Figure 2D, left panel). Immortalization is only obtained after transduction of both Cdk4 and hTERT; cells from both patients still undergo senescence with only hTERT or had a slightly extended lifespan with only Cdk4 (Figure 2A and B). Western blots show increased Cdk4 expression in HCEC CTs compared to noninfected HCECs, indicating successful integration and expression of Cdk4 (Figure 3A). Telomere restriction fragment length analysis assays show that the telomere lengths of nonimmortalized HCECs progressively shorten with increased PD (Figure 3B) and, while heterogeneous in length, the shortest telomeres range from 1.8 to 2.6 Kb at senescence. In contrast, the immortalized HCECs maintain telomere
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).
lengths well above 10 Kb. Telomerase activity (telomeric repeat amplification protocol) assays for the immortalized HCEC lines (HCEC 1CT and HCEC 2CT) show activity levels comparable to that in a control cell line (HeLa) indicating successful hTERT integration and expression (Figure 3C). In contrast, absence of the processive 6-bp ladders in the telomeric repeat amplification protocol assay in nonimmortalized HCEC1 and 2 parental cells indicates lack or very low levels of telomerase activity that are insufficient to maintain telomere length.
Characterization of Immortalized HCECs HCEC CTs cultured in 2% serum appear as compact cuboidal to spindle-shaped cells (Figure 4A). These
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cells are actively proliferating given a positive proliferating cell nuclear antigen status (Figure 4B) and some express various mesenchymal markers, such as vimentin (data not shown) and ␣-smooth muscle actin (Figure 4C). Placing cells in reduced growth factors and treating with GSK-3 inhibitors 6-bromoindirubin-3-oxime (BIO) or LiCl induces subconfluent HCEC CTs to display an increased epithelial cuboidal morphology (Figure 4D; yellow arrows indicating cells form circular structures with a central opening). Cells under this treatment are growth arrested (lack of proliferating cell nuclear antigen status
staining; Figure 4E) and no longer express the mesenchymal markers (Figure 4F). In this growth arrested state, cells exhibit various epithelial and intestinal/colonic specific markers. Cytokeratin 18 is visible in 30% of cells (Figure 5A). More than 90% of cells express a juxtanuclear pattern for cytokeratin 20, a pattern previously described in epithelial cells that are newly synthesizing intermediated filaments (Figure 5B).33 Zonula occludens-1 (ZO-1), a tight junction protein, and -catenin, a component of adherens junctions, are observed to localize to the cell membranes (Figure 5C and
Figure 4. Representative images characterizing HCEC CTs in log-growth and growth arresting conditions (BIO treated). (A) Appearance of HCEC CTs in loggrowth. (B, C) Positive expression of proliferating cell nuclear antigen (PCNA) status (green) and ␣-smooth muscle actin (␣-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 ␣-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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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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Figure 5. Expression of epithelial markers in growth arrested (BIO treated) cells (cytokeratin 18, cytokeratin 20, zonula occludens-1 [ZO-1], -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 m for all images.
D, respectively). The colon epithelial cell specific marker A33 antigen is observed in the cytoplasm and cell membranes in ⬃98% of the cells (Figure 5E). Villin is localized in a perinuclear and cytoplasmic distribution primarily in HCEC 1CT (Figure 5F), a pattern seen in colonic epithelial cells that are in an undifferentiated state.34 Mucin-1 (MUC1), a transmembrane mucin identified in human colonic crypts35,36 and proposed to be a marker of nonterminally differentiated colonic epithelial cells in the middle to bottom of colonic crypts,37 is observed in 70%⫺90% of cells, and has been confirmed by Western blot in both the growth arrested and log-phase growth cells (Figure 5G and Supplementary Figure 1; presence of multiple bands represents the protein with varying degrees of glycosylation products).38,39 In addition, approximately 15% of HCEC CT cells in the growth arrested state display faint perinuclear and cytoplasmic staining of the goblet cell marker MUC2 (Figure 5H). Approximately 20% of HCEC CTs stain for dipeptidyl peptidase 4, an enzyme found in the epithelium of the small intestine and colon that corresponds to the absorptive cell lineage (Figure 5I). Interestingly, approximately 6% of the population of cells stain positively for the neuroendocrine marker chromogranin A, though only in log-growth cells and not in the growth arrested state (Figure 5J).
Rhodamine and fluorescein isothiocyanate controls are shown in Figure 5K and 5L. Although immortalized HCECs from both patients displayed A33, MUC2, dipeptidyl peptidase 4, and chromogranin A on immunostaining, the signal intensity for all these antibodies was overall higher in HCEC 1CT compared to HCEC 2CT. Supplementary Figure 2 shows the percentages of cells from each patient expressing colonic epithelial markers. Evidence of wnt activity in cells was assessed via Western blots for -catenin in the nuclear and cytoplasmic compartments (Supplementary Figure 3). These results show detectable nuclear levels in both log-growth and growtharrested cells, with modest accumulation of -catenin in HCEC 1CT after treatment with BIO. In summary, these results demonstrate that in logphase growth, HCEC CTs share both epithelial and mesenchymal features. When growth is arrested, cells transition to a cuboidal morphology, the mesenchymal markers disappear, and markers corresponding to colonic epithelial cells become apparent.
HCECs Express Stem Cell Markers Given that HCEC CTs exhibit both epithelial and mesenchymal features, and that there is higher expression of stem cell markers in epithelial cells possessing
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HCEC CTs in monolayer culture have epithelial progenitor cell features.
Ultrastructural Studies Transmission electron microscopy was used to visualize the ultrastructure of one of the immortalized HCECs (HCECT 1CT). Proliferating cells have scarce organelles consistent with undifferentiated cells (Supplementary Figure 6). In monolayer cultures of growth arrested cells, there is still lack of clear microvilli and brush border again consistent with undifferentiated cells, but there is evidence of increased organelles (eg, Golgi, endoplasmic reticulum, mitochondria), suggesting increased synthesis of proteins (Supplementary Figure 7, right panel). There are also structures resembling tripartite junctions, composed of various electron dense intermembrane (desmosomal-like) complexes, adherens junctions, and an apical tight junction, observed to form in between adjacent cells (Supplementary Figure 7, left panel). Given the paucity of attaching intermediate filaments to the desmosomal structures, these are likely to be immature, but the presence of 8⫺10 nm diameter intermediate filaments stretching to the dense structure (arrowheads) indicates that these are likely to be desmosomes. JuncBASIC– ALIMENTARY TRACT
mesenchymal features,40 microarray analyses were used as an initial approach to characterize the HCEC CTs (Gene Expression Omnibus accession numbers: GSE 18433 and GSM459373-GSM459388). The results reveal that the HCEC 1CT and HCEC 2CT have relatively high mRNA expression of the stem cell markers CD29 (1-integrin), CD44, CD166, BMI1, survivin, and lower expression of LGR5 (Supplementary Figure 4).41,42 Staining with both mouse monoclonal and rabbit polyclonal LGR5 antibodies consistently revealed prominent expression of LGR5 in close to 100% of HCEC CTs, with HCEC 2CT showing lower expression than HCEC 1CT (Figure 6A and B). Human colon fibroblasts (C26Ci) do not show LGR5 staining under equal camera exposure times (Figure 6C). Significant expression was also observed for BMI1 in 90%⫺95% of both HCEC CT lines (Figure 6D and E). C26Ci fibroblasts, while showing some staining for BMI1, show decreased signal under equal camera exposure times compared to HCEC 1CT and to HCEC 2CT (Figure 6F). Expression of the stem cell markers CD29 and CD44 were validated via Western blots for both HCEC 1CT and 2CT in log-growth and growth arrested conditions (Supplementary Figure 5A and B; HCEC 2CT CD44 Western blot not shown). The combined presence of colonic epithelial and stem cell markers suggest that
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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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tions are seen in growth arrested cells and not in logphase growth cells.
HCEC CTs Proliferate Into Self-Organizing Cysts in Matrigel and Express Absorptive and Mucus-Secreting Markers
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We next addressed if HCEC CTs were capable of differentiating into various epithelial lineages of the colon (absorptive, mucus secreting, and neuroendocrine cells) in 3D organotypic culture in Matrigel. Populations of HCEC 2CTs were grown inside Matrigel in serumsupplemented media in the absence of feeder layers to assess for multipotent capacity. Subsets of individual cells proliferate and form multicellular structures (Figure 7A, days 1⫺6). The number of multicellular structures corresponds to about 10% of the seeded cells. Some of these structures continue to grow beyond 7 days and make larger cyst-like structures with a hollow interior. As visualized by confocal microscopy small multicellular structures (day 11) exhibit continuous ZO-1 staining surrounding and in between the cells (Figure 7B). Within the cytoplasm and circumscribed by the ZO-1, there is MUC2 staining (Figure 7C). At later time points (18 days) villin expression is observed lining the central lumen of the cyst-like structure, suggesting a polarized distribution (Figure 7D). MUC2 is observed in a subset of cells comprising the cysts (Figure 7E). These results suggest that individual cells are able to proliferate and give rise to the absorptive (villin expressing) and goblet cell (MUC2 expressing) lineages of the colon.
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Karyotype of Immortalized HCEC CTs and DNA Sequencing Analysis The karyotypes of HCEC CTs from both patients are diploid (Supplementary Figure 8A showing karyotype for HCEC 1CT, PD 90). DNA sequencing of mutational hotspots in APC, KRAS, and TP53 (genes important for colon cancer progression) revealed no amino acid changing mutations (data not shown). HCEC 1CT and 2CT do not display tumorigenic properties, such as anchorage independent growth (Supplementary Figure 8B, upper panel illustrating individual cells) compared to a colon cancer cell line HCT116 (Supplementary Figure 8B, lower panel illustrating large soft agar colonies of cells) or tumor formation in nude mice (data not shown).
Discussion To study the development of colonic diseases in vitro, such as colorectal cancer progression, culture models consisting of cytogenetically normal and nontumorigenic HCEC lines are needed. We show in this report that nontransformed epithelial cells extracted from colonic biopsies can be immortalized, maintained in culture as undifferentiated epithelial progenitor cells, and differentiate into more mature colonic epithelial cells when placed in a 3D environment. While it was formally possible that the mesenchymal and epithelial features present during log-phase growth immortalized colonic cells is due to a mixed population of epithelial cells and pericryptal fibroblasts, we think this is highly unlikely. For example, we have isolated
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.
multiple clones of these cells and these retain some mesenchymal markers when in logarithmic growth, which disappear when growth arrested and epithelial markers appear. The complete disappearance of mesenchymal morphology and markers, and the emergence of epithelial features in HCEC CTs after BIO treatment suggest other factors may explain the mixed cellular features observed in the population of immortalized cells. It is possible that the mesenchymal features in HCEC CTs may be a consequence of sustained growth in 2-dimensional (2D) culture. Previous reports describe the use of GSK-3 inhibitors (LiCl) and more highly specific inhibitors (BIO) to alter mesenchymal features and promote an epithelial state.43– 46 One possibility is that the 2D culture environment and/or other unidentified stressors promote colonic epithelial cells to assume mesenchymal features. It is known that components in serum, such as transforming growth factor⫺, can confer mesenchymal features to epithelial cells of various organs both in vitro and in vivo.47– 49 Other reports also describe how various nontransformed epithelial cell types, such as those from the small intestine, thyroid, liver, and lens downregulate epithelial markers and upregulate a mesenchymal phenotype in vitro in response to stress, the 2D tissue culture environment, or wound induced migration.46,50 –52 The effect of GSK-3 inhibitors on HCEC CTs in vitro possibly suggests an important role of downstream affected targets, such as Wnt signaling, in the promotion of an epithelial phenotype in normal colonic epithelial cells in vivo. HCEC CTs in 2D monolayer conditions exhibit markers consistent with the lineages of the colonic mucosa, yet they do not show features of the mature colonic epithelium, such as microvilli and a brush border. This undifferentiated phenotype in 2D culture may be a normal response of epithelial progenitor cells to being removed from the crypt microenvironment. For example, it is recognized that in the mammalian intestine and colon, proper growth, and differentiation of epithelial cells depends on a complex interplay of signaling cascades, presence of specific substrates and growth factors, and preserved interactions with neighboring mesenchymal cells.53,54 The ability of the HCEC CTs to exhibit features of differentiated colonic epithelial cells in 3D culture, such as apical villin, continuous ZO-1, and prominent MUC2 underscores the importance of an appropriate microenvironment in promoting and maintaining epithelial features of nontransformed human colonic epithelial cells. The multilineage differentiation capacity of individual HCEC CTs in a 3D culture highlights the potential role of intestinal and colonic early progenitor or stem markers in identifying undifferentiated human colonic progenitor epithelial cells with multipotent capacity. Although some of these markers have been better characterized in the small intestines and colons of mice (BMI1,
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LGR5), or shown to be present in the cells of the lower portions of human colonic crypts (MUC1, 1-integrin, CD44, survivin), their role as stem cell markers of normal human colonic epithelial cells is a matter of debate. It is interesting that BMI1 is present in actively proliferating HCECs because this marker has been observed in the compartment corresponding to the quiescent cells of mice small intestine. One possible explanation is that BMI1 expression may be a result of high p16 levels in the immortalized cells, given that BMI1 is a negative regulator of p16. Thus, BMI1 expression may be a result of cellular immortalization. This would not explain why comparable BMI1 levels are observed in the microarray studies in the early PD nonimmortalized cells. The fact that LGR5 is detected in replicating HCEC CTs is consistent with LGR5 being seen in actively replicating undifferentiated mouse crypt base cells. Although there is a lack of knowledge with respect to the role of recently identified gastrointestinal stem cell markers in human cells, their presence in the undifferentiated HCEC CTs and the capability of these cells to differentiate in 3D culture suggest a potential relevance of these markers to the undifferentiated precursor epithelial cells of human colonic crypts. In conclusion, we demonstrate the successful immortalization of nonneoplastic human colonic epithelial progenitor cells from 2 different patients. The cells are able to propagate in long-term culture and have been cryopreserved for future experimentation. In 2D culture, the cells have progenitor features, yet retain the ability to self-organize and differentiate in 3D Matrigel culture. These cells should serve as valuable reagents to study differentiation as well as to investigate the initiation and progression of colonic diseases, such as colon cancer.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2009.11.052. References 1. Buset M, Winawer S, Friedman E. Defining conditions to promote the attachment of adult human colonic epithelial cells. In Vitro Cell Dev Biol 1987;23:403– 412. 2. Whitehead RH, Brown A, Bhathal PS. A method for the isolation and culture of human colonic crypts in collagen gels. In Vitro Cell Dev Biol 1987;23:436 – 442. 3. Deveney CW, Rand-Luby L, Rutten MJ, et al. Establishment of human colonic epithelial cells in long-term culture. J Surg Res 1996;64:161–169. 4. Latella G, Fonti R, Caprilli R, et al. Characterization of the mucins produced by normal human colonocytes in primary culture. Int J Colorectal Dis 1996;11:76 – 83. 5. Pang G, Buret A, O’Loughlin E, et al. Immunologic, functional, and morphological characterization of three new human small intestinal epithelial cell lines. Gastroenterology 1996;111:8 –18.
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6. Quaroni A, Beaulieu JF. Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells. Gastroenterology 1997;113:1198 –1213. 7. Perreault N, Beaulieu JF. Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp Cell Res 1998; 245:34 – 42. 8. Panja A. A novel method for the establishment of a pure population of nontransformed human intestinal primary epithelial cell (HIPEC) lines in long term culture. Lab Invest 2000;80:1473– 1475. 9. Pedersen G, Saermark T, Giese B, et al. A simple method to establish short-term cultures of normal human colonic epithelial cells from endoscopic biopsy specimens. Comparison of isolation methods, assessment of viability and metabolic activity. Scand J Gastroenterol 2000;35:772–780. 10. Grossmann J, Walther K, Artinger M, et al. Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC). Eur J Cell Biol 2003;82: 262–270. 11. Packer L, Fuehr K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 1977;267:423– 425. 12. Ramirez RD, Morales CP, Herbert BS, et al. Putative telomereindependent mechanisms of replicative aging reflect inadequate growth conditions. Genes Dev 2001;15:398 – 403. 13. Forsyth NR, Evans AP, Shay JW, et al. Developmental differences in the immortalization of lung fibroblasts by telomerase. Aging Cell 2003;2:235–243. 14. Parrinello S, Samper E, Krtolica A, et al. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 2003;5:741–747. 15. Bodnar AG, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998;279:349 –352. 16. Ramirez RD, Herbert BS, Vaughan MB, et al. Bypass of telomeredependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 2003;22:433– 444. 17. Ramirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 2004;64:9027–9034. 18. Morales CP, Holt SE, Ouellette M, et al. Absence of cancerassociated changes in human fibroblasts immortalized with telomerase. Nat Genet 1999;21:115–118. 19. Barker N, van Es JH, Kuipers J, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007; 449:1003–1007. 20. Becker L, Huang Q, Mashimo H. Immunostaining of Lgr5, an intestinal stem cell marker, in normal and premalignant human gastrointestinal tissue. Sci World J 2008;8:1168 –1176. 21. Samuel S, Walsh R, Webb J, et al. Characterization of putative stem cells in isolated human colonic crypt epithelial cells and their interactions with myofibroblasts. Am J Physiol Cell Physiol 2009;296:C296 –C305. 22. Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 2008;40:915–920. 23. Reinisch C, Kandutsch S, Uthman A, et al. BMI-1: a protein expressed in stem cells, specialized cells and tumors of the gastrointestinal tract. Histol Histopathol 2006;21:1143–1149. 24. Fujimoto K, Beauchamp RD, Whitehead RH. Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression. Gastroenterology 2002;123: 1941–1948. 25. Huang EH, Hynes MJ, Zhang T, et al. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 2009;69:3382–3389.
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26. Ootani A, Li X, Sangiorgi E, et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med 2009;15:701–706. 27. Sato T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009;459:262–265. 28. Forsyth NR, Morales CP, Damle S, et al. Spontaneous immortalization of clinically normal colon-derived fibroblasts from a familial adenomatous polyposis patient. Neoplasia 2004;6:258 –265. 29. Booth C, O’Shea JA. Isolation and culture of intestinal and epithelial cells. In: Freshney RI, FM, ed. Culture of epithelial cells. 2nd ed. New York: Wiley-Liss, 2002:303–337. 30. Evans GS, Flint N, Somers AS, et al. The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J Cell Sci 1992;101:219 –231. 31. Wright WE, Shay JW. Inexpensive low-oxygen incubators. Nat Protoc 2006;1:2088 –2090. 32. Ince TA, Richardson AL, Bell GW, et al. Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes. Cancer Cell 2007;12:160 –170. 33. Blouin R, Kawahara H, French SW, et al. Selective accumulation of IF proteins at a focal juxtanuclear site in COS-1 cells transfected with mouse keratin 18 cDNA. Exp Cell Res 1990;187: 234 –242. 34. Dudouet B, Robine S, Huet C, et al. Changes in villin synthesis and subcellular distribution during intestinal differentiation of HT29-18 clones. J Cell Biol 1987;105:359 –369. 35. Byrd JC, Bresalier RS. Mucins and mucin binding proteins in colorectal cancer. Cancer Metastasis Rev 2004;23:77–99. 36. Cao Y, Blohm D, Ghadimi BM, et al. Mucins (MUC1 and MUC3) of gastrointestinal and breast epithelia reveal different and heterogeneous tumor-associated aberrations in glycosylation. J Histochem Cytochem 1997;45:1547–1557. 37. Ajioka Y, Allison LJ, Jass JR. Significance of MUC1 and MUC2 mucin expression in colorectal cancer. J Clin Pathol 1996;49: 560 –564. 38. Burke PA, Gregg JP, Bakhtiar B, et al. Characterization of MUC1 glycoprotein on prostate cancer for selection of targeting molecules. Int J Oncol 2006;29:49 –55. 39. Tytgat KM, Swallow DM, Van Klinken BJ, et al. Unpredictable behaviour of mucins in SDS/polyacrylamide-gel electrophoresis. Biochem J 1995;310:1053–1054. 40. Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704 –715. 41. Dalerba P, Dylla SJ, Park IK, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A 2007;104:10158 –10163. 42. Kim PJ, Plescia J, Clevers H, et al. Survivin and molecular pathogenesis of colorectal cancer. Lancet 2003;362:205–209. 43. Davies JA, Garrod DR. Induction of early stages of kidney tubule differentiation by lithium ions. Dev Biol 1995;167:50 – 60. 44. Kuure S, Popsueva A, Jakobson M, et al. Glycogen synthase kinase-3 inactivation and stabilization of beta-catenin induce nephron differentiation in isolated mouse and rat kidney mesenchymes. J Am Soc Nephrol 2007;18:1130 –1139. 45. Meijer L, Skaltsounis AL, Magiatis P, et al. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 2003;10: 1255–1266. 46. Stump RJ, Lovicu FJ, Ang SL, et al. Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition. J Pathol 2006;210: 249 –257. 47. Boland S, Boisvieux-Ulrich E, Houcine O, et al. TGF beta 1 promotes actin cytoskeleton reorganization and migratory phenotype
48.
49.
50.
51.
52.
53.
in epithelial tracheal cells in primary culture. J Cell Sci 1996; 109:2207–2219. Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 2003;112:1776 – 1784. Miettinen PJ, Ebner R, Lopez AR, et al. TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J Cell Biol 1994; 127:2021–2036. Godoy P, Hengstler JG, Ilkavets I, et al. Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology 2009;49:2031–2043. Ulianich L, Garbi C, Treglia AS, et al. ER stress is associated with dedifferentiation and an epithelial-to-mesenchymal transition-like phenotype in PC Cl3 thyroid cells. J Cell Sci 2008;121:477– 486. Znalesniak EB, Durer U, Hoffmann W. Expression profiling of stationary and migratory intestinal epithelial cells after in vitro wounding: restitution is accompanied by cell differentiation. Cell Physiol Biochem 2009;24:125–132. Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet 2006;7:349 –359.
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54. Auclair BA, Benoit YD, Rivard N, et al. Bone morphogenetic protein signaling is essential for terminal differentiation of the intestinal secretory cell lineage. Gastroenterology 2007;133:887– 896.
Received August 19, 2009. Accepted November 20, 2009. Reprint requests Address requests for reprints to: Jerry W. Shay, PhD, Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390. e-mail: [email protected]; fax: (214) 648-8694. Acknowledgments We thank Ying Zou for the in situ assays and initial karyotyping. Conflicts of interest The authors disclose no conflicts. Funding This work was supported by NASA grant no. NNXO8BA54G and NNX09AU95G to J.W.S., and T32 CA124334 and American Gastroenterology Association Fellow to Faculty Transition Award to A.I.R.
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Supplementary Materials and Methods Conditions to Induce Growth Arrest of Colonic Epithelial Progenitor Cells Immortalized cells seeded at a density of 5.0 ⫻ 103 on Deckglasser cover-slips (Mülheim, Germany) inside 24-well Falcon plastic dishes (BD Biosciences) were allowed to proliferate until subconfluent and then exposed to lithium chloride (LiCl 30 mM; Sigma) for 5 days or 6-bromoindirubin-3-oxime (BIO 5.0 M; Calbiochem, San Diego, CA) for 3 days in HCEC supplemented X media with the serum decreased to 0.1% and epithelial growth factor (EGF) to 1.25 ng/mL. Depending on the primary antibody to be used for staining, cells were fixed with either a 1:1 mixture of cold methanol and acetone or neutral buffered formalin after BIO exposure and stored in 4°C until staining.
Telomeric Repeat Amplification Protocol, Western Blotting, and Immunocytochemistry Telomeric repeat amplification assay was performed as described previously.1 Primary antibodies used for Western blot and immunostaining were mouse antibodies against Cdk-4, p16INK4A (both from Millipore, Billerica, MA), MUC1 (BD Biosciences), CD29, CD44 (both from Santa Cruz Biotechnology, Santa Cruz, CA) and anti–actin (Sigma). For immunofluorescence staining, primary antibodies used were mouse monoclonal antibodies against cytokeratin 18 (Santa Cruz Biotechnology), chromogranin A (Santa Cruz Biotechnology), ␣-smooth muscle actin (Sigma), vimentin (Millipore), -catenin (Abcam, Cambridge, MA), villin (BD Biosciences), BMI1 (Abcam), MUC1 (BD Biosciences); rabbit polyclonal antibody against ZO-1 (Zymed Laboratories, South San Francisco, CA), A33 (Santa Cruz Biotechnology), LGR5 (GenTex, Irvine, CA), MUC2 (Santa Cruz Biotechnology), proliferating cell nuclear antigen (PCNA; Abcam), cytokeratin 20 (Santa Cruz Biotechnology), and rabbit monoclonal against GPR49 (LGR5) (Abcam). With the exception of BMI1 and MUC1, cells on coverslips were fixed with a 1:1 mixture of cold acetone and methanol for 5 minutes. For BMI1 and MUC1 staining cells were fixed with neutral buffered formalin for 10 minutes. Species-specific goat rhodamine or fluorescein isothiocyanate–labeled antibodies (Invitrogen, Carlsbad, CA) were used for secondary antibody staining. A drop of Vectashield with DAPI (Vector Laboratories, Burlingame, CA) was
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added to coverslips and these were subsequently mounted onto glass slides for visualization with an epifluorescence microscope (Zeiss Axiovert 200M).
Tumorigenic Assays For growth in soft agar, 1000 cells per well were suspended in 0.37% Noble agar (Difco, BD Biosciences) in supplemented X media and overlaid over 0.5% Noble agar in triplicate 12-well plates. The number of macroscopically visible colonies was counted after 3–5 weeks of growth. For assessment of tumor formation in nude mice 5 ⫻ 106 cells were injected subcutaneously and maintained in a germ-free barrier for up to 6 months or until tumor size required euthanasia.
Electron Microscopy Cells were seeded onto Thermanox plastic coverslips (Nalge Nunc International, Rochester, NY). Logphase and growth-arrested immortalized cells were fixed with glutaraldehyde and tannic acid for 20 minutes, embedded in Epon resin and visualized with a Tecnai G2 Spirit 120KV transmission electron microscope.
Matrigel Culture After immortalized cell cultures were trypsinized into a single-cell suspension, between 200 and 1000 cells were mixed with 300 L undiluted Matrigel (BD Biosciences) and seeded on 24-well plate dishes. For immunostaining experiments, 500 cells were mixed with 50 uL Matrigel and seeded on top of Fisherbrand microscope cover glasses (Fisher Scientific, Pittsburgh, PA). The coverslips with Matrigel were submerged under 0.5 mL supplemented X media for up 18 days. In situ processing for immunofluorescence staining was performed as described previously.2 A Zeiss Axiovert 200M epifluorescence microscope and Leica TCS SP5 confocal microscope were used to visualize organoids. Supplementary References 1. Ramirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 2004;64:9027–9034. 2. Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in threedimensional basement membrane cultures. Methods 2003;30: 256 –268.
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Cytoplasmic Fraction HCEC2CT Bio
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Beta-catenin
Beta actin 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).
Nuclear Fraction Bio
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Beta-catenin MRE-11 Supplementary Figure 3. Western blots demonstrating cytoplasmic and nuclear -catenin levels in log-growth and growth-arrested (BIO) cells.
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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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. 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.
gr ow re th du ce d se re ru du m ce /E d G se F, ru Lo no m g /E BI gr G O ow F, re t + h du BI ce O d se re ru du m ce /E d G se F, ru no m /E BI G O F, + BI O
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HCEC 1CT 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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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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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.
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,* and JERRY W. SHAY* *Department of Cell Biology; ‡Department of Medicine, Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas; and § Veterans Affairs North Texas Health Care System, Dallas, Texas
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BACKGROUND & AIMS: Long-term propagation of human colonic epithelial cells (HCEC) of adult origin has been a challenge; currently used HCEC lines are of malignant origin and/or contain multiple cytogenetic changes. We sought to immortalize human colon biopsyderived cells expressing stem cell markers and retaining multilineage epithelial differentiation capability. METHODS: We isolated and cultured cells from biopsy samples of 2 patients undergoing routine screening colonoscopy. Cells were immortalized by expression of the nononcogenic proteins cyclin-dependent kinase 4 (Cdk4) and the catalytic component of human telomerase (hTERT) and maintained for more than 1 year in culture. RESULTS: The actively proliferating HCECs expressed the mesenchymal markers vimentin and ␣-smooth muscle actin. Upon growth arrest, cells assumed a cuboidal shape, decreased their mesenchymal features, and expressed markers of colonic epithelial cells such as cytokeratin 18, zonula occludens-1, mucins-1 and -2, antigen A33, and dipeptidyl peptidase 4. Immortalized cells expressed stem cell markers that included LGR5, BMI1, CD29, and CD44. When placed in Matrigel in the absence of a mesenchymal feeder layer, individual cells divided and formed self-organizing, cyst-like structures; a subset of cells exhibited mucin-2 or polarized villin staining. CONCLUSIONS: We established immortalized HCECs that are capable of self-renewal and multilineage differentiation. These cells should serve as valuable reagents for studying colon stem cell biology, differentiation, and pathogenesis. Keywords: Stem/Progenitor Cells; Epithelial Mesenchymal Transition; Self-Renewal; Multilineage Differentiation.
T
he long-term propagation of human colonic epithelial cells (HCEC) of adult origin has historically been challenging. Although progress has taken place in the development of methods to culture HCECs, including the description of some long-term culture models, these techniques and cell lines have not gained wide acceptance in the scientific community possibly as a result of inherent technical difficulties in extracting and maintaining viable cells.1–10 A majority of the currently used HCEC lines are of malignant origin and/or contain multiple
cytogenetic changes. Although these cell lines have been highly informative in many aspects of intestinal cell biology research, established adult HCEC lines not derived from tumor specimens and that are karyotypically normal are needed to conduct research that would be more difficult to perform with the currently established cell lines. For example, cell culture models of nontransformed adult HCECs could be used to test current paradigms of colorectal cancer progression. Creating de novo genetic changes, via a candidate or screening approach, would permit validation of known or novel mutations that confer a growth advantage to cells. This could then serve as a basis for the development and application of agents to test on cells, to determine if they have an effect on the aberrant pathways leading to transformation, and potentially permit the discovery of new drugs that mitigate colorectal cancer development. In the present studies, we demonstrate the long-term growth of adult human colonic epithelial progenitor cells extracted from colonic biopsy specimens taken from patients undergoing screening colonoscopy. We show that in low-oxygen culture conditions, cells replicate and can be immortalized with the nononcogenic proteins cyclindependent kinase 4 ([Cdk4], bypasses cell culture associated stresses that frequently lead to premature senescence) and the catalytic component of the human ribonucleoprotein enzyme telomerase ([hTERT]; maintains telomere length and prevents replicative senescence). Low oxygen tension (2%⫺5% oxygen) has previously been shown to extend the replicative lifespan of many human cell types (fibroblasts and epithelial cells) by delaying the stress-induced senescence that results from normal atmospheric (21%) oxygen tension.11–14 Cdk4 and hTERT have previously been used to immortalize various epithelial cell types without conferring tuAbbreviations used in this paper: BIO, bromoindirubin-3-oxime; 2D, two-dimensional; 3D, three-dimensional; Cdk4, cyclin-dependent kinase-4; EGF, epithelial growth factor; GSK-3, glycogen synthase kinase-3; HCEC CT, human colonic epithelial cell immortalized with Cdk4 and hTERT; hTERT, catalytic component of human telomerase; MUC, mucin; PD, population doublings; ZO-1, zonula occludens-1. © 2010 by the AGA Institute 0016-5085/10/$36.00 doi:10.1053/j.gastro.2009.11.052
morigenic properties.15–18 Characterization of the immortalized colonic cells reveals that they exhibit morphological features and markers characteristic of epithelial cells, particularly when growth is arrested as a result from serum deprivation, reduced epithelial growth factor (EGF), and treatment with glycogen synthase kinase-3  (GSK-3) inhibitors. In addition, the cells display LGR5 and BMI1, proteins marking the stem cells of the mice gastrointestinal tract and that also have been identified in the cells of human colonic crypts,19 –23 as well as other proteins marking the rapidly proliferating human colonic epithelial cells in vivo (CD29 and CD44).24,25 Importantly, individual immortalized cells can proliferate and subsequently differentiate into multicellular structures and express multilineage colonic differentiation specific markers, similar to self-organizing structures formed by mouse intestinal epithelial cells in 3-dimensional (3D) culture.26,27
Methods Colonic Tissues This study was approved by the Institutional Review Board at the Dallas VA Medical Center. Colon biopsies (20⫺30 samples, ⬃0.5 cm3) from tissue not involved with endoscopically visible adenomas were obtained from patients undergoing routine screening colonoscopy after obtaining informed consent.
Growth Media and Tissue Culture Substrate Cells were routinely grown on basal X media (HyClone, Logan, UT) supplemented with EGF (25 ng/ mL) (PeproTech, Inc, Rocky Hill, NJ), hydrocortisone (1 g/mL), insulin (10 g/mL), transferrin (2 g/mL), sodium selenite (5 nanomolar) (all from Sigma, St Louis, MO), 2% cosmic calf serum (HyClone), and gentamicin sulfate (50 g/mL) (Gemini Bio-Products, West Sacramento, CA). Cells were cultured in Primaria flasks (BD Biosciences, San Jose, CA) and grown in 2%⫺5% oxygen and 7% carbon dioxide. HCT 116, BJ human skin fibroblasts, HeLa cells, and immortalized human colonic fibroblast cells (C26Ci, population doubling 150)28 were maintained in X media supplemented with 10% cosmic calf serum (HyClone). C26Ci cells were treated with 10 g/mL mitomycin C (Sigma) for 2 hours and used as feeder layers from the point of initial crypt attachment until the first passage.
Cell Isolation and Immortalization The intestinal cell isolation techniques previously described were used with some modifications.29,30 Colonic biopsies were immersed in cold X medium, brought to the laboratory within 40⫺60 minutes after colonoscopy, copiously washed with phosphate-buffered saline containing antibiotic/antimycotic solution (Gemini BioProducts), and cut into multiple small pieces ( ⬃1 mm in
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size). After enzymatic digestion with collagenase 150 U/mL (Worthington Biochemical, Lakewood, NJ) and dispase 40 g/mL (Roche, Germany), crypts were resuspended in X medium with growth supplements including 2% serum, and plated in Primaria culture dishes seeded 48 hours previously with 50% confluent colonic fibroblast feeder layers. During the first 10 days after attachment, cells were fed every 3 days, reducing the serum by 1% each change until 0% to prevent growth of unwanted cells such as fibroblasts. Once small nests of expanding epithelial cells were easily observed, cells were transduced with retroviral Cdk4 and hTERT as described previously.17 When numerous cuboidal-appearing cell nests were observed (3⫺4 weeks after initial crypt seeding), cells were reseeded on Primaria flasks. Feeder layers were not needed or used for routine tissue culture after the first passage.
Growth Arrest Conditions, Telomeric Repeat Amplification Protocol, Western Blotting, Immunocytochemistry, Tumorigenic Assays, Electron Microscopy, Matrigel Culture Please refer to Supplementary Materials and Methods section for these standard procedures.
Results Primary Culture and Immortalization of Cells Figure 1 describes the crypt extraction procedure. Enzymatic digestion yielded a higher number of viable crypts compared with chelation methods. Initial expansion of cells is limited in normoxic/atmospheric (21%) conditions and in conventional plastic culture dishes. Thus, optimized culture conditions include low oxygen tension (2%⫺5% O2),31 modified substrate (Primaria plastic culture dishes32), and initial growth of cells in half confluent human colon fibroblast-feeder layers. After the first passage, cells are subcultured in the presence of 2% serum, but in the absence of feeder layers. Cells from these primary cultures can continuously divide in these conditions for approximately 40 population doublings (PD) (Figure 2A and B). Cells obtained from the 2 patient specimens were transduced with both Cdk4 and hTERT. The resulting immortalized cells are termed human colonic epithelial cell (HCEC) 1CT (patient 1; “C” for Cdk4 and “T” for telomerase) and HCEC 2CT (patient 2). Both lines have been continuously subcultured for ⬎100 PD, are able to be cryopreserved, and have been reintroduced into cell cultures without complications (Figure 2A and B). Cessation of growth of nonimmortalized cells is accompanied by a flattened and enlarged morphological phenotype typical of cells undergoing senescence (Figure 2C, right panel). Compared to the flattened and enlarged shape of the nonimmortalized controls at senescence, the compact appearance of immortalized HCEC CTs PD 90 (Figure 2D, right panel) is similar to the morphology of
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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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earlier passaged cells in log-phase growth (Figure 2D, left panel). Immortalization is only obtained after transduction of both Cdk4 and hTERT; cells from both patients still undergo senescence with only hTERT or had a slightly extended lifespan with only Cdk4 (Figure 2A and B). Western blots show increased Cdk4 expression in HCEC CTs compared to noninfected HCECs, indicating successful integration and expression of Cdk4 (Figure 3A). Telomere restriction fragment length analysis assays show that the telomere lengths of nonimmortalized HCECs progressively shorten with increased PD (Figure 3B) and, while heterogeneous in length, the shortest telomeres range from 1.8 to 2.6 Kb at senescence. In contrast, the immortalized HCECs maintain telomere
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).
lengths well above 10 Kb. Telomerase activity (telomeric repeat amplification protocol) assays for the immortalized HCEC lines (HCEC 1CT and HCEC 2CT) show activity levels comparable to that in a control cell line (HeLa) indicating successful hTERT integration and expression (Figure 3C). In contrast, absence of the processive 6-bp ladders in the telomeric repeat amplification protocol assay in nonimmortalized HCEC1 and 2 parental cells indicates lack or very low levels of telomerase activity that are insufficient to maintain telomere length.
Characterization of Immortalized HCECs HCEC CTs cultured in 2% serum appear as compact cuboidal to spindle-shaped cells (Figure 4A). These
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cells are actively proliferating given a positive proliferating cell nuclear antigen status (Figure 4B) and some express various mesenchymal markers, such as vimentin (data not shown) and ␣-smooth muscle actin (Figure 4C). Placing cells in reduced growth factors and treating with GSK-3 inhibitors 6-bromoindirubin-3-oxime (BIO) or LiCl induces subconfluent HCEC CTs to display an increased epithelial cuboidal morphology (Figure 4D; yellow arrows indicating cells form circular structures with a central opening). Cells under this treatment are growth arrested (lack of proliferating cell nuclear antigen status
staining; Figure 4E) and no longer express the mesenchymal markers (Figure 4F). In this growth arrested state, cells exhibit various epithelial and intestinal/colonic specific markers. Cytokeratin 18 is visible in 30% of cells (Figure 5A). More than 90% of cells express a juxtanuclear pattern for cytokeratin 20, a pattern previously described in epithelial cells that are newly synthesizing intermediated filaments (Figure 5B).33 Zonula occludens-1 (ZO-1), a tight junction protein, and -catenin, a component of adherens junctions, are observed to localize to the cell membranes (Figure 5C and
Figure 4. Representative images characterizing HCEC CTs in log-growth and growth arresting conditions (BIO treated). (A) Appearance of HCEC CTs in loggrowth. (B, C) Positive expression of proliferating cell nuclear antigen (PCNA) status (green) and ␣-smooth muscle actin (␣-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 ␣-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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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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Figure 5. Expression of epithelial markers in growth arrested (BIO treated) cells (cytokeratin 18, cytokeratin 20, zonula occludens-1 [ZO-1], -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 m for all images.
D, respectively). The colon epithelial cell specific marker A33 antigen is observed in the cytoplasm and cell membranes in ⬃98% of the cells (Figure 5E). Villin is localized in a perinuclear and cytoplasmic distribution primarily in HCEC 1CT (Figure 5F), a pattern seen in colonic epithelial cells that are in an undifferentiated state.34 Mucin-1 (MUC1), a transmembrane mucin identified in human colonic crypts35,36 and proposed to be a marker of nonterminally differentiated colonic epithelial cells in the middle to bottom of colonic crypts,37 is observed in 70%⫺90% of cells, and has been confirmed by Western blot in both the growth arrested and log-phase growth cells (Figure 5G and Supplementary Figure 1; presence of multiple bands represents the protein with varying degrees of glycosylation products).38,39 In addition, approximately 15% of HCEC CT cells in the growth arrested state display faint perinuclear and cytoplasmic staining of the goblet cell marker MUC2 (Figure 5H). Approximately 20% of HCEC CTs stain for dipeptidyl peptidase 4, an enzyme found in the epithelium of the small intestine and colon that corresponds to the absorptive cell lineage (Figure 5I). Interestingly, approximately 6% of the population of cells stain positively for the neuroendocrine marker chromogranin A, though only in log-growth cells and not in the growth arrested state (Figure 5J).
Rhodamine and fluorescein isothiocyanate controls are shown in Figure 5K and 5L. Although immortalized HCECs from both patients displayed A33, MUC2, dipeptidyl peptidase 4, and chromogranin A on immunostaining, the signal intensity for all these antibodies was overall higher in HCEC 1CT compared to HCEC 2CT. Supplementary Figure 2 shows the percentages of cells from each patient expressing colonic epithelial markers. Evidence of wnt activity in cells was assessed via Western blots for -catenin in the nuclear and cytoplasmic compartments (Supplementary Figure 3). These results show detectable nuclear levels in both log-growth and growtharrested cells, with modest accumulation of -catenin in HCEC 1CT after treatment with BIO. In summary, these results demonstrate that in logphase growth, HCEC CTs share both epithelial and mesenchymal features. When growth is arrested, cells transition to a cuboidal morphology, the mesenchymal markers disappear, and markers corresponding to colonic epithelial cells become apparent.
HCECs Express Stem Cell Markers Given that HCEC CTs exhibit both epithelial and mesenchymal features, and that there is higher expression of stem cell markers in epithelial cells possessing
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HCEC CTs in monolayer culture have epithelial progenitor cell features.
Ultrastructural Studies Transmission electron microscopy was used to visualize the ultrastructure of one of the immortalized HCECs (HCECT 1CT). Proliferating cells have scarce organelles consistent with undifferentiated cells (Supplementary Figure 6). In monolayer cultures of growth arrested cells, there is still lack of clear microvilli and brush border again consistent with undifferentiated cells, but there is evidence of increased organelles (eg, Golgi, endoplasmic reticulum, mitochondria), suggesting increased synthesis of proteins (Supplementary Figure 7, right panel). There are also structures resembling tripartite junctions, composed of various electron dense intermembrane (desmosomal-like) complexes, adherens junctions, and an apical tight junction, observed to form in between adjacent cells (Supplementary Figure 7, left panel). Given the paucity of attaching intermediate filaments to the desmosomal structures, these are likely to be immature, but the presence of 8⫺10 nm diameter intermediate filaments stretching to the dense structure (arrowheads) indicates that these are likely to be desmosomes. JuncBASIC– ALIMENTARY TRACT
mesenchymal features,40 microarray analyses were used as an initial approach to characterize the HCEC CTs (Gene Expression Omnibus accession numbers: GSE 18433 and GSM459373-GSM459388). The results reveal that the HCEC 1CT and HCEC 2CT have relatively high mRNA expression of the stem cell markers CD29 (1-integrin), CD44, CD166, BMI1, survivin, and lower expression of LGR5 (Supplementary Figure 4).41,42 Staining with both mouse monoclonal and rabbit polyclonal LGR5 antibodies consistently revealed prominent expression of LGR5 in close to 100% of HCEC CTs, with HCEC 2CT showing lower expression than HCEC 1CT (Figure 6A and B). Human colon fibroblasts (C26Ci) do not show LGR5 staining under equal camera exposure times (Figure 6C). Significant expression was also observed for BMI1 in 90%⫺95% of both HCEC CT lines (Figure 6D and E). C26Ci fibroblasts, while showing some staining for BMI1, show decreased signal under equal camera exposure times compared to HCEC 1CT and to HCEC 2CT (Figure 6F). Expression of the stem cell markers CD29 and CD44 were validated via Western blots for both HCEC 1CT and 2CT in log-growth and growth arrested conditions (Supplementary Figure 5A and B; HCEC 2CT CD44 Western blot not shown). The combined presence of colonic epithelial and stem cell markers suggest that
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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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tions are seen in growth arrested cells and not in logphase growth cells.
HCEC CTs Proliferate Into Self-Organizing Cysts in Matrigel and Express Absorptive and Mucus-Secreting Markers
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We next addressed if HCEC CTs were capable of differentiating into various epithelial lineages of the colon (absorptive, mucus secreting, and neuroendocrine cells) in 3D organotypic culture in Matrigel. Populations of HCEC 2CTs were grown inside Matrigel in serumsupplemented media in the absence of feeder layers to assess for multipotent capacity. Subsets of individual cells proliferate and form multicellular structures (Figure 7A, days 1⫺6). The number of multicellular structures corresponds to about 10% of the seeded cells. Some of these structures continue to grow beyond 7 days and make larger cyst-like structures with a hollow interior. As visualized by confocal microscopy small multicellular structures (day 11) exhibit continuous ZO-1 staining surrounding and in between the cells (Figure 7B). Within the cytoplasm and circumscribed by the ZO-1, there is MUC2 staining (Figure 7C). At later time points (18 days) villin expression is observed lining the central lumen of the cyst-like structure, suggesting a polarized distribution (Figure 7D). MUC2 is observed in a subset of cells comprising the cysts (Figure 7E). These results suggest that individual cells are able to proliferate and give rise to the absorptive (villin expressing) and goblet cell (MUC2 expressing) lineages of the colon.
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Karyotype of Immortalized HCEC CTs and DNA Sequencing Analysis The karyotypes of HCEC CTs from both patients are diploid (Supplementary Figure 8A showing karyotype for HCEC 1CT, PD 90). DNA sequencing of mutational hotspots in APC, KRAS, and TP53 (genes important for colon cancer progression) revealed no amino acid changing mutations (data not shown). HCEC 1CT and 2CT do not display tumorigenic properties, such as anchorage independent growth (Supplementary Figure 8B, upper panel illustrating individual cells) compared to a colon cancer cell line HCT116 (Supplementary Figure 8B, lower panel illustrating large soft agar colonies of cells) or tumor formation in nude mice (data not shown).
Discussion To study the development of colonic diseases in vitro, such as colorectal cancer progression, culture models consisting of cytogenetically normal and nontumorigenic HCEC lines are needed. We show in this report that nontransformed epithelial cells extracted from colonic biopsies can be immortalized, maintained in culture as undifferentiated epithelial progenitor cells, and differentiate into more mature colonic epithelial cells when placed in a 3D environment. While it was formally possible that the mesenchymal and epithelial features present during log-phase growth immortalized colonic cells is due to a mixed population of epithelial cells and pericryptal fibroblasts, we think this is highly unlikely. For example, we have isolated
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.
multiple clones of these cells and these retain some mesenchymal markers when in logarithmic growth, which disappear when growth arrested and epithelial markers appear. The complete disappearance of mesenchymal morphology and markers, and the emergence of epithelial features in HCEC CTs after BIO treatment suggest other factors may explain the mixed cellular features observed in the population of immortalized cells. It is possible that the mesenchymal features in HCEC CTs may be a consequence of sustained growth in 2-dimensional (2D) culture. Previous reports describe the use of GSK-3 inhibitors (LiCl) and more highly specific inhibitors (BIO) to alter mesenchymal features and promote an epithelial state.43– 46 One possibility is that the 2D culture environment and/or other unidentified stressors promote colonic epithelial cells to assume mesenchymal features. It is known that components in serum, such as transforming growth factor⫺, can confer mesenchymal features to epithelial cells of various organs both in vitro and in vivo.47– 49 Other reports also describe how various nontransformed epithelial cell types, such as those from the small intestine, thyroid, liver, and lens downregulate epithelial markers and upregulate a mesenchymal phenotype in vitro in response to stress, the 2D tissue culture environment, or wound induced migration.46,50 –52 The effect of GSK-3 inhibitors on HCEC CTs in vitro possibly suggests an important role of downstream affected targets, such as Wnt signaling, in the promotion of an epithelial phenotype in normal colonic epithelial cells in vivo. HCEC CTs in 2D monolayer conditions exhibit markers consistent with the lineages of the colonic mucosa, yet they do not show features of the mature colonic epithelium, such as microvilli and a brush border. This undifferentiated phenotype in 2D culture may be a normal response of epithelial progenitor cells to being removed from the crypt microenvironment. For example, it is recognized that in the mammalian intestine and colon, proper growth, and differentiation of epithelial cells depends on a complex interplay of signaling cascades, presence of specific substrates and growth factors, and preserved interactions with neighboring mesenchymal cells.53,54 The ability of the HCEC CTs to exhibit features of differentiated colonic epithelial cells in 3D culture, such as apical villin, continuous ZO-1, and prominent MUC2 underscores the importance of an appropriate microenvironment in promoting and maintaining epithelial features of nontransformed human colonic epithelial cells. The multilineage differentiation capacity of individual HCEC CTs in a 3D culture highlights the potential role of intestinal and colonic early progenitor or stem markers in identifying undifferentiated human colonic progenitor epithelial cells with multipotent capacity. Although some of these markers have been better characterized in the small intestines and colons of mice (BMI1,
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LGR5), or shown to be present in the cells of the lower portions of human colonic crypts (MUC1, 1-integrin, CD44, survivin), their role as stem cell markers of normal human colonic epithelial cells is a matter of debate. It is interesting that BMI1 is present in actively proliferating HCECs because this marker has been observed in the compartment corresponding to the quiescent cells of mice small intestine. One possible explanation is that BMI1 expression may be a result of high p16 levels in the immortalized cells, given that BMI1 is a negative regulator of p16. Thus, BMI1 expression may be a result of cellular immortalization. This would not explain why comparable BMI1 levels are observed in the microarray studies in the early PD nonimmortalized cells. The fact that LGR5 is detected in replicating HCEC CTs is consistent with LGR5 being seen in actively replicating undifferentiated mouse crypt base cells. Although there is a lack of knowledge with respect to the role of recently identified gastrointestinal stem cell markers in human cells, their presence in the undifferentiated HCEC CTs and the capability of these cells to differentiate in 3D culture suggest a potential relevance of these markers to the undifferentiated precursor epithelial cells of human colonic crypts. In conclusion, we demonstrate the successful immortalization of nonneoplastic human colonic epithelial progenitor cells from 2 different patients. The cells are able to propagate in long-term culture and have been cryopreserved for future experimentation. In 2D culture, the cells have progenitor features, yet retain the ability to self-organize and differentiate in 3D Matrigel culture. These cells should serve as valuable reagents to study differentiation as well as to investigate the initiation and progression of colonic diseases, such as colon cancer.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2009.11.052. References 1. Buset M, Winawer S, Friedman E. Defining conditions to promote the attachment of adult human colonic epithelial cells. In Vitro Cell Dev Biol 1987;23:403– 412. 2. Whitehead RH, Brown A, Bhathal PS. A method for the isolation and culture of human colonic crypts in collagen gels. In Vitro Cell Dev Biol 1987;23:436 – 442. 3. Deveney CW, Rand-Luby L, Rutten MJ, et al. Establishment of human colonic epithelial cells in long-term culture. J Surg Res 1996;64:161–169. 4. Latella G, Fonti R, Caprilli R, et al. Characterization of the mucins produced by normal human colonocytes in primary culture. Int J Colorectal Dis 1996;11:76 – 83. 5. Pang G, Buret A, O’Loughlin E, et al. Immunologic, functional, and morphological characterization of three new human small intestinal epithelial cell lines. Gastroenterology 1996;111:8 –18.
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6. Quaroni A, Beaulieu JF. Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells. Gastroenterology 1997;113:1198 –1213. 7. Perreault N, Beaulieu JF. Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp Cell Res 1998; 245:34 – 42. 8. Panja A. A novel method for the establishment of a pure population of nontransformed human intestinal primary epithelial cell (HIPEC) lines in long term culture. Lab Invest 2000;80:1473– 1475. 9. Pedersen G, Saermark T, Giese B, et al. A simple method to establish short-term cultures of normal human colonic epithelial cells from endoscopic biopsy specimens. Comparison of isolation methods, assessment of viability and metabolic activity. Scand J Gastroenterol 2000;35:772–780. 10. Grossmann J, Walther K, Artinger M, et al. Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC). Eur J Cell Biol 2003;82: 262–270. 11. Packer L, Fuehr K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 1977;267:423– 425. 12. Ramirez RD, Morales CP, Herbert BS, et al. Putative telomereindependent mechanisms of replicative aging reflect inadequate growth conditions. Genes Dev 2001;15:398 – 403. 13. Forsyth NR, Evans AP, Shay JW, et al. Developmental differences in the immortalization of lung fibroblasts by telomerase. Aging Cell 2003;2:235–243. 14. Parrinello S, Samper E, Krtolica A, et al. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 2003;5:741–747. 15. Bodnar AG, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998;279:349 –352. 16. Ramirez RD, Herbert BS, Vaughan MB, et al. Bypass of telomeredependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 2003;22:433– 444. 17. Ramirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 2004;64:9027–9034. 18. Morales CP, Holt SE, Ouellette M, et al. Absence of cancerassociated changes in human fibroblasts immortalized with telomerase. Nat Genet 1999;21:115–118. 19. Barker N, van Es JH, Kuipers J, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007; 449:1003–1007. 20. Becker L, Huang Q, Mashimo H. Immunostaining of Lgr5, an intestinal stem cell marker, in normal and premalignant human gastrointestinal tissue. Sci World J 2008;8:1168 –1176. 21. Samuel S, Walsh R, Webb J, et al. Characterization of putative stem cells in isolated human colonic crypt epithelial cells and their interactions with myofibroblasts. Am J Physiol Cell Physiol 2009;296:C296 –C305. 22. Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 2008;40:915–920. 23. Reinisch C, Kandutsch S, Uthman A, et al. BMI-1: a protein expressed in stem cells, specialized cells and tumors of the gastrointestinal tract. Histol Histopathol 2006;21:1143–1149. 24. Fujimoto K, Beauchamp RD, Whitehead RH. Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression. Gastroenterology 2002;123: 1941–1948. 25. Huang EH, Hynes MJ, Zhang T, et al. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 2009;69:3382–3389.
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26. Ootani A, Li X, Sangiorgi E, et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med 2009;15:701–706. 27. Sato T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009;459:262–265. 28. Forsyth NR, Morales CP, Damle S, et al. Spontaneous immortalization of clinically normal colon-derived fibroblasts from a familial adenomatous polyposis patient. Neoplasia 2004;6:258 –265. 29. Booth C, O’Shea JA. Isolation and culture of intestinal and epithelial cells. In: Freshney RI, FM, ed. Culture of epithelial cells. 2nd ed. New York: Wiley-Liss, 2002:303–337. 30. Evans GS, Flint N, Somers AS, et al. The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J Cell Sci 1992;101:219 –231. 31. Wright WE, Shay JW. Inexpensive low-oxygen incubators. Nat Protoc 2006;1:2088 –2090. 32. Ince TA, Richardson AL, Bell GW, et al. Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes. Cancer Cell 2007;12:160 –170. 33. Blouin R, Kawahara H, French SW, et al. Selective accumulation of IF proteins at a focal juxtanuclear site in COS-1 cells transfected with mouse keratin 18 cDNA. Exp Cell Res 1990;187: 234 –242. 34. Dudouet B, Robine S, Huet C, et al. Changes in villin synthesis and subcellular distribution during intestinal differentiation of HT29-18 clones. J Cell Biol 1987;105:359 –369. 35. Byrd JC, Bresalier RS. Mucins and mucin binding proteins in colorectal cancer. Cancer Metastasis Rev 2004;23:77–99. 36. Cao Y, Blohm D, Ghadimi BM, et al. Mucins (MUC1 and MUC3) of gastrointestinal and breast epithelia reveal different and heterogeneous tumor-associated aberrations in glycosylation. J Histochem Cytochem 1997;45:1547–1557. 37. Ajioka Y, Allison LJ, Jass JR. Significance of MUC1 and MUC2 mucin expression in colorectal cancer. J Clin Pathol 1996;49: 560 –564. 38. Burke PA, Gregg JP, Bakhtiar B, et al. Characterization of MUC1 glycoprotein on prostate cancer for selection of targeting molecules. Int J Oncol 2006;29:49 –55. 39. Tytgat KM, Swallow DM, Van Klinken BJ, et al. Unpredictable behaviour of mucins in SDS/polyacrylamide-gel electrophoresis. Biochem J 1995;310:1053–1054. 40. Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704 –715. 41. Dalerba P, Dylla SJ, Park IK, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A 2007;104:10158 –10163. 42. Kim PJ, Plescia J, Clevers H, et al. Survivin and molecular pathogenesis of colorectal cancer. Lancet 2003;362:205–209. 43. Davies JA, Garrod DR. Induction of early stages of kidney tubule differentiation by lithium ions. Dev Biol 1995;167:50 – 60. 44. Kuure S, Popsueva A, Jakobson M, et al. Glycogen synthase kinase-3 inactivation and stabilization of beta-catenin induce nephron differentiation in isolated mouse and rat kidney mesenchymes. J Am Soc Nephrol 2007;18:1130 –1139. 45. Meijer L, Skaltsounis AL, Magiatis P, et al. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 2003;10: 1255–1266. 46. Stump RJ, Lovicu FJ, Ang SL, et al. Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition. J Pathol 2006;210: 249 –257. 47. Boland S, Boisvieux-Ulrich E, Houcine O, et al. TGF beta 1 promotes actin cytoskeleton reorganization and migratory phenotype
48.
49.
50.
51.
52.
53.
in epithelial tracheal cells in primary culture. J Cell Sci 1996; 109:2207–2219. Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 2003;112:1776 – 1784. Miettinen PJ, Ebner R, Lopez AR, et al. TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J Cell Biol 1994; 127:2021–2036. Godoy P, Hengstler JG, Ilkavets I, et al. Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology 2009;49:2031–2043. Ulianich L, Garbi C, Treglia AS, et al. ER stress is associated with dedifferentiation and an epithelial-to-mesenchymal transition-like phenotype in PC Cl3 thyroid cells. J Cell Sci 2008;121:477– 486. Znalesniak EB, Durer U, Hoffmann W. Expression profiling of stationary and migratory intestinal epithelial cells after in vitro wounding: restitution is accompanied by cell differentiation. Cell Physiol Biochem 2009;24:125–132. Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet 2006;7:349 –359.
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54. Auclair BA, Benoit YD, Rivard N, et al. Bone morphogenetic protein signaling is essential for terminal differentiation of the intestinal secretory cell lineage. Gastroenterology 2007;133:887– 896.
Received August 19, 2009. Accepted November 20, 2009. Reprint requests Address requests for reprints to: Jerry W. Shay, PhD, Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390. e-mail: [email protected]; fax: (214) 648-8694. Acknowledgments We thank Ying Zou for the in situ assays and initial karyotyping. Conflicts of interest The authors disclose no conflicts. Funding This work was supported by NASA grant no. NNXO8BA54G and NNX09AU95G to J.W.S., and T32 CA124334 and American Gastroenterology Association Fellow to Faculty Transition Award to A.I.R.
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Supplementary Materials and Methods Conditions to Induce Growth Arrest of Colonic Epithelial Progenitor Cells Immortalized cells seeded at a density of 5.0 ⫻ 103 on Deckglasser cover-slips (Mülheim, Germany) inside 24-well Falcon plastic dishes (BD Biosciences) were allowed to proliferate until subconfluent and then exposed to lithium chloride (LiCl 30 mM; Sigma) for 5 days or 6-bromoindirubin-3-oxime (BIO 5.0 M; Calbiochem, San Diego, CA) for 3 days in HCEC supplemented X media with the serum decreased to 0.1% and epithelial growth factor (EGF) to 1.25 ng/mL. Depending on the primary antibody to be used for staining, cells were fixed with either a 1:1 mixture of cold methanol and acetone or neutral buffered formalin after BIO exposure and stored in 4°C until staining.
Telomeric Repeat Amplification Protocol, Western Blotting, and Immunocytochemistry Telomeric repeat amplification assay was performed as described previously.1 Primary antibodies used for Western blot and immunostaining were mouse antibodies against Cdk-4, p16INK4A (both from Millipore, Billerica, MA), MUC1 (BD Biosciences), CD29, CD44 (both from Santa Cruz Biotechnology, Santa Cruz, CA) and anti–actin (Sigma). For immunofluorescence staining, primary antibodies used were mouse monoclonal antibodies against cytokeratin 18 (Santa Cruz Biotechnology), chromogranin A (Santa Cruz Biotechnology), ␣-smooth muscle actin (Sigma), vimentin (Millipore), -catenin (Abcam, Cambridge, MA), villin (BD Biosciences), BMI1 (Abcam), MUC1 (BD Biosciences); rabbit polyclonal antibody against ZO-1 (Zymed Laboratories, South San Francisco, CA), A33 (Santa Cruz Biotechnology), LGR5 (GenTex, Irvine, CA), MUC2 (Santa Cruz Biotechnology), proliferating cell nuclear antigen (PCNA; Abcam), cytokeratin 20 (Santa Cruz Biotechnology), and rabbit monoclonal against GPR49 (LGR5) (Abcam). With the exception of BMI1 and MUC1, cells on coverslips were fixed with a 1:1 mixture of cold acetone and methanol for 5 minutes. For BMI1 and MUC1 staining cells were fixed with neutral buffered formalin for 10 minutes. Species-specific goat rhodamine or fluorescein isothiocyanate–labeled antibodies (Invitrogen, Carlsbad, CA) were used for secondary antibody staining. A drop of Vectashield with DAPI (Vector Laboratories, Burlingame, CA) was
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added to coverslips and these were subsequently mounted onto glass slides for visualization with an epifluorescence microscope (Zeiss Axiovert 200M).
Tumorigenic Assays For growth in soft agar, 1000 cells per well were suspended in 0.37% Noble agar (Difco, BD Biosciences) in supplemented X media and overlaid over 0.5% Noble agar in triplicate 12-well plates. The number of macroscopically visible colonies was counted after 3–5 weeks of growth. For assessment of tumor formation in nude mice 5 ⫻ 106 cells were injected subcutaneously and maintained in a germ-free barrier for up to 6 months or until tumor size required euthanasia.
Electron Microscopy Cells were seeded onto Thermanox plastic coverslips (Nalge Nunc International, Rochester, NY). Logphase and growth-arrested immortalized cells were fixed with glutaraldehyde and tannic acid for 20 minutes, embedded in Epon resin and visualized with a Tecnai G2 Spirit 120KV transmission electron microscope.
Matrigel Culture After immortalized cell cultures were trypsinized into a single-cell suspension, between 200 and 1000 cells were mixed with 300 L undiluted Matrigel (BD Biosciences) and seeded on 24-well plate dishes. For immunostaining experiments, 500 cells were mixed with 50 uL Matrigel and seeded on top of Fisherbrand microscope cover glasses (Fisher Scientific, Pittsburgh, PA). The coverslips with Matrigel were submerged under 0.5 mL supplemented X media for up 18 days. In situ processing for immunofluorescence staining was performed as described previously.2 A Zeiss Axiovert 200M epifluorescence microscope and Leica TCS SP5 confocal microscope were used to visualize organoids. Supplementary References 1. Ramirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 2004;64:9027–9034. 2. Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in threedimensional basement membrane cultures. Methods 2003;30: 256 –268.
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Cytoplasmic Fraction HCEC2CT Bio
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Beta actin 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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Beta-catenin MRE-11 Supplementary Figure 3. Western blots demonstrating cytoplasmic and nuclear -catenin levels in log-growth and growth-arrested (BIO) cells.
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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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. 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.
gr ow re th du ce d se re ru du m ce /E d G se F, ru Lo no m g /E BI gr G O ow F, re t + h du BI ce O d se re ru du m ce /E d G se F, ru no m /E BI G O F, + BI O
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HCEC 1CT 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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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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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.