In vivo gene expression profiling of human intestinal epithelial cells: analysis by laser microdissection of formalin fixed tissues
© George et al; licensee BioMed Central Ltd. 2008
Received: 21 November 2007
Accepted: 05 May 2008
Published: 05 May 2008
The small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However, the differences in transcriptional programming and the molecular machinery that governs the migration, adhesion, and differentiation of intestinal epithelial cell lineages in humans remain under-explored. To increase our understanding of these mechanisms, we have evaluated gene expression patterns of ileal epithelial cells isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of healthy human small intestinal mucosa. Expression profiles in villus and crypt epithelium were determined by DNA microarray, quantitative real-time PCR, and immunohistochemistry based methods. The expression levels of selected epithelial biomarkers were also compared between gastrointestinal tissues.
Previously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations (GEO accession: GSE10629). Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5) and regenerating islet-derived 3 alpha (Reg3A), were detected exclusively within the small bowel crypt, most notably in the ileum in comparison to other sites along the gastrointestinal tract.
The elucidation of differential gene expression patterns between crypt and villus epithelial cell lineages in human ileal tissue provides novel insights into the molecular machinery that mediates their functions and spatial organization. Moreover, our findings establish an important framework of knowledge for future investigations of human gastrointestinal diseases.
Epithelial stem cell progenitors, which reside in the intestinal crypt compartment, differentiate into either secretory (goblet, enteroendocrine, Paneth) or absorptive (enterocytes) lineages, that in turn, provide a range of digestive and host defense functions within the gastrointestinal tract. Studies suggest that epithelial cell maturation, differentiation, and turn-over occur through a complex orchestration of cellular adhesion and dissociation, migration, and programmed cell death [1, 2]. These events, and digestive and defense functions of the maturing cells, are likely accompanied and/or driven by wholesale alterations in gene expression. Three of the epithelial cell types migrate upward along the villus – absorptive enterocytes, goblet cells, and the far less abundant enteroendocrine cells. Absorptive enterocytes perform their nutrient absorptive and digestive functions via nutrient transport proteins and hydrolytic enzymes [3–5]. Goblet cells perform cytoprotective, anti-inflammatory, and maintenance functions through synthesis and secretion of trefoil factors and mucins [6, 7]. Enteroendocrine cells are individually dispersed neuroendocrine cells that represent less than 1% of the gut epithelial cells and produce a variety of gut hormones . Paneth cells, on the other hand, remain in the crypt and reside at the base, where they provide innate antimicrobial functions through production and secretion of antibiotics that combat invasion by potential pathogens and may help regulate the composition of commensal bacteria [9–12].
A range of human diseases and digestive disorders have been linked to disruptions in the proper maturation and/or terminal function of these epithelial lineages, highlighting the need to increase our knowledge of their basic physiology. Biochemical, genetic, and gene expression studies in mouse models and in vitro epithelial cell lines have, in recent years, provided insights towards understanding both the roles of Wnt signaling in mediating proliferation of intestinal stem cell populations [13, 14], and of Wnt and Notch signaling in the differentiation of secretory populations [15–17]. Several biomarkers believed to mediate differentiation of progenitor stem cells have also been identified [18–21]. However, many details regarding the mechanisms that underlie intestinal epithelial cell differentiation and the coordination of interactions with adjacent cells and the extracellular matrix remain unknown. Moreover, similarities and differences between human intestinal epithelial cell differentiation and events observed in the mouse models and in various epithelial cell lines have not been fully established.
To address these needs, we have evaluated gene expression patterns in human ileal epithelial cells isolated by laser capture microdissection (LCMD) from either the villus or crypt epithelial cell regions of the crypt-villus axis. Genes differentially expressed in the two cellular compartments were identified through high throughput genome-scale microarray analysis and further assessed by quantitative real-time PCR and immunohistochemistry. Biomarkers previously identified in vitro and in animal models as either Paneth cell or villus epithelial cell specific were found to be enriched in the corresponding two cell populations in the human small intestine. Additionally, a distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation was found to be differentially transcribed in each population, providing novel insights into potentially important details about the molecular machinery ileal epithelial cells utilize to mediate their spatial organization and function.
Differential expression profiling of villus and crypt epithelial cell lineages
Supportive evidence for microarray findings
Transcriptional profiling of ileal crypt epithelium
Increased resistin-like beta (RETNLB) transcription was also detected in crypt epithelial cells (Figure 6B). In the colon, RETNLB is synthesized in both goblet and crypt region epithelial cells and appears to govern susceptibility to colonic inflammation . Previous studies also suggest that RETNLB may regulate expression of Reg3A . Thus, the coincident high level expression of RETNLB and Reg3A in crypt cells indicates that these molecules may function coordinately in the human ileum to mediate homeostasis of the commensal microbiota and/or attenuate inflammatory responses.
Transcriptional programs in villus epithelial cells
Transcriptional patterns of ileal epithelial biomarkers along the GI tract
Interestingly, we found that Paneth cell markers HD5 and Reg3A were highly expressed in the ileum and moderately expressed in the jejunum, but not in the colon, or esophagus. A low level of mRNA, well below ileal and jejunal levels, was also detected in the duodenum, indicating that these antimicrobial peptides may function primarily in the more distal regions of the small bowel, consistent with the distribution of Paneth cells in the intestinal tract. In contrast, RETNLB and EphRB3 were transcribed predominantly at other sites, including the colon (RETNLB and EphRB3) and esophagus (EphRB3 only).
While LCMD and gene expression analyses have been employed successfully in studies of the adult mouse model and intestinal epithelial cell lines to identify mechanisms of function, differentiation, and positioning [3, 4, 19, 21, 28], to our knowledge the current report represents the first such in vivo investigation of the human small intestinal crypt-villus axis. It is notable, therefore, that many of the patterns of transcription observed in the villus and crypt epithelium pools that we identified here were in agreement with data from these previous reports. The consistency of these data with previous studies both underscored the reproducibility of the methodologies used for LCMD and mRNA detection in paraformaldehyde-fixed samples, and supported the overall accuracy of transcriptome data generated by microarray analysis. While it is likely that the integrity of mRNA isolated from paraformaldehyde fixed specimens is inferior to fresh-frozen specimens, we have found that short, overnight fixation protocols yield RNA of sufficient quality to perform consistent and reproducible RT-PCR and microarray analyses. However, due to the small sample sizes generated by LCMD, it was not feasible to check the RNA integrity by gel methods before proceeding to amplification and labeling. Thus, although the data presented here serves to illustrate the general feasibility and robustness of LCMD-based studies of paraformaldehyde fixed tissues, it remains possible that some small, but significant, differences between two cell pools may not be optimally detected by these methods and criteria.
Although high baseline transcription levels of alpha-defensins, lysozyme, and secretory phospholipase A2 in mammalian small intestinal Paneth cells have been documented [11, 33, 34], we were intrigued by the apparently similar level of transcription of the multifunctional lectin-related protein Reg3A. Reg3A transcription also displayed a markedly similar tissue specific expression profile to HD5 (Figure 5B), apparently transcribed along the alimentary tract exclusively within the small bowel. Like HD5 , Reg3A is known as a downstream Wnt/beta-catenin signaling target . The murine ortholog of Reg3A (named Reg3γ in mice), is expressed in Paneth cells . While this localization is notable in light of studies demonstrating its antimicrobial properties [23, 36], Reg3A has also been shown to stimulate liver regeneration and growth . Thus, it is possible that in the small intestine, high basal levels of Reg3A expression may act bifunctionally to contribute to rapid and robust local antimicrobial functions when required, and perhaps to promote epithelial cell differentiation or growth.
Potentially important cellular pathways employed by Paneth cells to enable rapid secretion of antimicrobials may have been discovered within the transcriptional profiles of the crypt epithelium, including enriched expression of the Ras-like GTP binding protein, Rab26. Although little is yet known about Rab26 function, the few studies that have been completed suggest it may be involved in mediating the transport of secretory granules in parotid acinar cells of the rat [38, 39]. These investigations showed that Rab26 is exclusively associated with mature secretory granules that are recruited to the plasma membrane in response to increased Ca++ influx induced by beta-adrenergic stimulation. Given these findings, we suggest that Rab26 may function similarly in ileal crypts, where release of secretory granules rich in antimicrobial peptides is a predominant physiological event.
Absorptive enterocytes and goblet cells are the predominant cell types of the villus epithelium (Figures 1A and 4). Our observation of considerable goblet cell numbers lining the villus epithelium in the ileal tissue samples utilized for LCMD (Fig 1) was reflected by increased mRNA levels of mucin 2 and mucin 3B (supplemental_microarray_data.xls). Among the transport associated genes we found enriched in the villus epithelium were several members of the solute carrier family (supplemental_microarray_data.xls). It is notable that this list included the oligopeptide transporter SLC15A1 and the amino acid transporter SLC7A7, both recognized as villus specific biomarkers, and shown to be expressed at high levels in the mouse ileum . While much of the previous data on SLC15A1 and SLC7A7 have been generated from transcriptional profiling of whole tissue to determine the relative distribution of SLC transporters along the gastrointestinal tract, our findings extend these studies to confirm the presence of these molecules in the absorptive epithelial regions of the human ileum. In addition to SLC15A1 and SLC7A7, we detected enriched expression of the sodium/hydrogen exchanger SLC9A3 and the sulfate transporter SLC26A2 in the villus epithelial cell pool. These findings are potentially important in understanding how different absorptive/digestive processes may be organized along specific regions of the human small intestine, and their relationship to similar processes in animal models.
Villus epithelial cells also displayed clear increases in the expression of several growth and differentiation associated genes, and genes involved in establishing and maintaining epithelial cell polarity, as compared to the levels observed in crypt epithelial cells. Of the genes involved in epithelial growth, it was interesting to note that many may function through EGF signaling; a pathway enriched in the microarray data (Figure 2). Among the enriched genes in the villus epithelium controlling growth and differentiation, Reg4 was particularly interesting due to its role in activating the EGF receptor , and its classification, like Paneth cell Reg3A, within the regenerating (Reg) family of genes . Thus, the large discrepancy between mRNA levels of Reg4 in the villus epithelium and Reg3A in crypt epithelium highlights that differential expression of this gene family may serve as useful indicators (perhaps even important mediators) of differentiation in human ileal epithelial cell lineages.
Our study provides a comprehensive evaluation of gene expression patterns that characterize villus and crypt epithelial cells in healthy human ileal tissue. In addition to detecting biomarkers previously identified in vitro and in animal models, we have gained insight into the predominate physiological processes occurring in these regions and identified a distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation in each population. As such, these data provide a framework upon which future investigations of human small intestinal epithelial development and function can be further explored. Moreover, improved knowledge of biomarkers specific to human epithelial cell lineages in the ileum will also aid in the design and interpretation of studies on enteric diseases, including infectious enteritis and Crohn's disease. In addition, the ability to accurately detect and analyze gene expression in isolated cell populations from formalin-fixed specimens represents a potentially valuable new tool to further our understanding of intestinal epithelial cell differentiation and function beyond the scope of animal models.
Surgical specimens of intestinal mucosa were obtained from individuals undergoing surgery for colon cancer, bowel obstruction, or other non-inflammatory conditions. These samples were obtained through the Cooperative Human Tissue Network (funded by the National Cancer Institute) with Institutional Review Board approval, and in accordance with the Helsinki Declaration. Other investigators may have received specimens from the same subjects. Immediately after surgery, the mucosa was separated from the adjacent tissue. The specimens were free of macroscopic signs of disease or inflammation. The mucosal tissue was either snap frozen in liquid nitrogen and stored at -80°C for subsequent RNA isolation, or fixed overnight in 4% (w/v) paraformaldehyde, dehydrated in a graded alcohol series and paraffin-embedded.
Cell isolations by LCMD
Epithelial cells from the terminal ileum were captured by LCMD from unstained 6 μm thick paraformaldehyde-fixed paraffin-embedded sections. All sectioning and subsequent specimen handling procedures were conducted under RNase free conditions. Sectioning was performed within two weeks of the LCMD procedure and cells were captured within one hour of deparafination. At least 500 epithelial cells of each type from each sample were laser catapulted using a PALM MicroLaser System (Zeiss, Oberkochen, Germany) into microcentrifuge tubes containing RNA extraction buffer. Total RNA was immediately extracted for microarray-based and real-time PCR-based gene expression analysis. All solutions and plasticware for these procedures were documented to be free of RNases.
RNA isolation and reverse transcription for PCR analysis
Frozen tissue specimens were disrupted mechanically in guanidine thiocyanate buffer, and total RNA was isolated using cesium chloride gradient ultracentrifugation as described . RNA was quantitated in duplicate using ultraviolet absorption spectrometry at 260 nm (Nanodrop Technologies, Wilmington, DE). For cDNA synthesis, 5 μg of total RNA was reverse transcribed with Superscript II reverse transcriptase (50 units, Invitrogen, Carlsbad, CA) using an oligo-(dT)12–18 primer as described . The single-stranded cDNA product was purified using column adsorption chromatography (Qiagen, Valencia, CA). A single cDNA preparation from each specimen was used for each assay. Alternatively, for 3'-rapid amplification of cDNA end (RACE) analysis 5 μg of total RNA from human small intestine was reverse transcribed using the RACE/anchor primer 5'-CCATCCTAATACGACTCACTATAGGGCTCGAGCGGC(T)18(GCA)(GCAT)-3' as described previously . The resulting cDNA product was used as a template in a PCR reaction using as gene specific primer common to both Reg3A and Reg3g 5'-CGCTGTCCCAAAGGCTCCAAG-3', and the antisense RACE primer 5'-CCATCCTAATACGACTCACTATAGGGC-3'. The reaction product was isolated and then subcloned. Ten independent RACE clones were isolated and DNA sequence analysis showed all corresponded to Reg3A.
Quantitative real-time PCR
Oligonucleotide sequences used in quantitative real-time RT-PCR analysis.
PCR Primer Pairs
5' TGACGCTCAGCAAAACCGATAAC 3'
5' AACAGGCTTCAGGACCAAGACAGC 3'
5' ACTCATCAATGTTGGGTCCTGTGG 3'
5' AGGGTCATCGTCACTTAGGCTTCC 3'
5' AACTCACCCAGCAACTCAATGCC 3'
5' CTCCTTCCCAATCTCCTCCTTCAG 3'
5' GCTGGTCAGAGACGGCAGAAAAC 3'
5' TGCTCCCTCCCTTTGTCCTGTAG 3'
5' AACGATGGGCAGTTCACGGTC 3'
5' GCTGTTGACAAGGATGTTGCGAG 3'
5' TGCTATCAACCTTTGCCCTCTACC 3'
5' CCCCAGAAGAACCCGTGTGAAC 3'
5' TGATGGTGGGCATGGGTCAG 3'
5' CGTGCTCGATGGGGTACTTCAG 3'
Expression of HD5 and Aldolase-B was detected, at the protein level, by immunohistochemistry analysis using mouse monoclonal antibodies to HD5  and aldolase-B (Dako, Carpinteria, CA). Sections were deparaffinized, hydrated in a graded series of alcohols, and washed in PBS pH 7.4. The sections were blocked with 5% normal goat serum, and then incubated in the mouse monoclonal antibody (dil 1:100) for 16 hours at 4°C in a humidified chamber. Sections were again washed in PBS, blocked with goat serum, followed by incubation with biotinylated goat anti-mouse (dil 1:200; Vector Laboratories, Inc., Burlingame, CA) for 20 minutes at room temperature. 5% normal goat serum was used as diluent for antisera. The sections were then treated with streptavidin-HRP complex and incubated with DAB substrate (Dako, Carpinteria, CA) for 8 minutes at room temperature, followed by light hematoxylin counterstain.
Amplification of mRNA was performed utilizing protocols and reagents in the Paradise Reagent System (Molecular Devices, Inc. Sunnyvale, CA). Labeling, hybridization to Human X3P Array GeneChips© (Affymetrix, Santa Clara, CA) staining and scanning were performed according to protocols in the Affymetrix Gene Expression Analysis Technical Manual. Labeled cRNA was hybridized for 16 hr at 45°C. The fluorescence emitted at 570 nm was measured and used to calculate and compare the expression level of each gene.
Stringent statistical criteria was applied to the analysis of microarray data using robust multi-chip analysis (RMA) based (Array Assist©) and model-based (dChip) algorithms . A minimum 2-fold difference between mean levels of transcription in villus and crypt epithelial cells was used as criteria for determining "enriched" expression in each population. Fold differences were calculated from comparison of the mean transcription levels among at least 3 biological replicate samples (patients) of each epithelial region, with 95% confidence (P-value ≤ 0.05) by standard student t-test. Genes meeting fold-change statistical criteria were subjected to hierarchical clustering to identify common patterns of up- and down-regulated transcription, followed by comprehensive functional and statistical analysis of the biological pathways and processes represented by the genes in each sub-cluster. The Affymetrix GeneChip Operating System data files (*.cel, and *.chp) have been deposited in and made available to the public at the Gene Expression Omnibus database (accession number: GSE10629).
Biofunctional analysis of microarray data
Assignment of genes to functional categories was performed through annotation of gene lists using the Affymetrix NetAFFX web interface, the DAVID annotation and biological process analysis tool , and through literature-based classification manually. Statistically over-represented (Fisher exact probability score < 0.05) biological processes within sub-clusters were identified using DAVID and Pathway Architect (Stratagne/Ioboin) analysis software. Following elucidation of biologically enriched themes, the microarray data was re-mined and analyzed with focus on identifying and interpreting all statistically valid changes gene expression associated with the highlighted pathways.
This work was supported by Public Health Service grants AI032738, AI43274, DK61297, and R21DE018097 from the National Institutes of Health. The authors thank Mr. Marko Estrada for excellent technical assistance with LCMD, RNA extraction, and cDNA synthesis.
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