BACKGROUND
epithelial stem cell progenitors, which reside in the intestinal crypt compartment, differentiate into either secretory  or absorptive  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  <cit> .
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  <cit> .
goblet cells perform cytoprotective, anti-inflammatory, and maintenance functions through synthesis and secretion of trefoil factors and mucins  <cit> .
enteroendocrine cells are individually dispersed neuroendocrine cells that represent less than 1% of the gut epithelial cells and produce a variety of gut hormones  <cit> .
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  <cit> .
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  <cit> , and of wnt and notch signaling in the differentiation of secretory populations  <cit> .
several biomarkers believed to mediate differentiation of progenitor stem cells have also been identified  <cit> .
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  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.
RESULTS
differential expression profiling of villus and crypt epithelial cell lineages
to elucidate differential gene expression profiles of small intestinal villus and crypt epithelial cells, these cell populations were excised from unstained, sectioned formalin-fixed human ileal tissue samples by lcmd .
the human ileal epithelial layer consists of multiple cell types  that simultaneously differentiate from stem cell progenitors as they migrate toward their sites of function along the villus-crypt axis.
villus epithelial cells were identified in unstained specimens based on topographical location; crypt epithelial cells were identified based on topographical location and appearance of granular cytoplasm characteristic of paneth cells.
following rna extraction, linear amplification, labeling, and hybridization to dna microarrays, the genes for which the level of detected transcripts differed by at least 2-fold  between the two cell pools were identified .
these genes were then hierarchically clustered to identify common patterns of differential expression .
genes that were expressed predominantly in either villus or crypt epithelia were then statistically evaluated to determine which pathways and processes were most represented .
genes associated with protein biosynthesis, cellular stress and antimicrobial responses, phosphorylation, and cell cycle  were enhanced in the crypt epithelial cells, while villus epithelial cells displayed increased expression of genes associated with lipid and carboxylic acid metabolism, transport, egf-like activity, and extracellular matrix functions.
a list of the genes differentially transcribed in these categories, their fold differences in expression between crypt and villus epithelial cells, and the statistical confidence associated with those differences, is provided in additional file .
the entire microarray data set is available for viewing at the gene expression omnibus database .
as expected, crypt epithelial cells expressed dramatically higher levels of antimicrobial peptides characteristic of paneth cells, including alpha-defensin- <dig> , hd <dig>  lysozyme, secretory phospholipase a <dig>  and reg3a .
race-pcr analysis confirmed that the alpha, and not the gamma, isoform of reg <dig> was expressed in the human ileal tissue , which contrasts with the nomenclature used in the mouse where reg3γ is expressed  <cit> .
trypsin, the proteolytic enzyme associated with alpha-defensin processing  <cit>  was also identified in the crypt.
villus epithelial cells, in contrast, displayed markedly increased mrna levels of aldolase-b, apolipoprotein-a <dig>  aminopeptidase-n, pep carboxykinase, and carboxylesterase- <dig>  the identification of these previously established intestinal epithelial biomarkers provided evidence that our isolation of the desired epithelial lineages was successful, and validated the reproducibility of the methodology using formalin-fixed paraffin-embedded samples across multiple biological replicates.
supportive evidence for microarray findings
to provide further support that the transcriptional profiles detected by microarray analysis accurately represented the differences between villus and crypt epithelial cells, we evaluated expression of selected genes by immunohistochemistry.
in agreement with transcriptome assessments and previous studies  <cit> , we detected the antimicrobial peptide hd <dig> exclusively in paneth cells within crypt regions .
aldolase-b, associated with carbohydrate metabolism in contrast, was detected primarily in epithelial cells lining the upper villus region within the same tissue sections .
these data served to both substantiate the corresponding microarray findings at the protein level, and provide further support for our interpretations of the global transcriptome data for crypt and villus epithelial cell populations.
to further test if the microarray analysis accurately represented the differences in mrna levels between villus and crypt epithelial cells, we evaluated expression of four genes by rt-pcr using the cdna pools obtained by lcmd.
expression of hd <dig> and reg3a mrna was readily detected in the crypt epithelial cell cdna pool, but was not evident in the villus epithelial cell cdna under the same conditions .
by contrast, aldolase b was detected in the villus epithelial cell cdna sample   <dig> cycles sooner than in the crypt epithelial cell cdna sample, suggesting a difference in mrna levels of approximately  <dig> ×  <dig> fold.
as a control, β-actin was detected in both cdna pools at approximately the same levels.
these data are consistent with the differential expression patterns of these genes detected by microarray hybridization analysis.
transcriptional profiling of ileal crypt epithelium
to elucidate potential mechanisms of paneth cell function, we evaluated candidate genes from the microarray data that were enriched in the crypt epithelial cell pool and believed to be associated with immune responses, cellular growth, differentiation, and motility.
one striking example, reg3a was transcribed, on average, at > <dig> fold higher levels in crypt epithelial cells than measured in villus epithelial cells .
reg3a has been shown previously to be expressed in intestinal paneth cells  <cit>  and has been shown to have both anti-inflammatory and antimicrobial properties  <cit> .
our findings indicate that reg3a is constitutively transcribed at substantial levels, similar to hd <dig> and trypsin in paneth cells of the human ileum.
these data suggest that in the healthy human ileum, reg3a may function predominantly in regulating bacterial colonization.
increased resistin-like beta  transcription was also detected in crypt epithelial cells .
in the colon, retnlb is synthesized in both goblet and crypt region epithelial cells and appears to govern susceptibility to colonic inflammation  <cit> .
previous studies also suggest that retnlb may regulate expression of reg3a  <cit> .
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.
other genes transcribed preferentially in crypt epithelial cells included several involved in epithelial growth and positioning that may represent a distinct set of biomarkers specific to this cellular compartment .
these included eph receptor b <dig>  homeobox b <dig>  and semaphorin 3f, implicated in previous studies to be involved in either the migration or specification of intestinal epithelial stem cell population.
ephrin b <dig> and its receptor are believed to be regulated by wnt and expressed in crypt epithelia where they control the proper segregation of paneth cells from post-mitotic cells  <cit> .
homeobox b <dig>  has been shown to regulate enteric development  <cit> , while several investigations have highlighted the role of semaphorin 3f in inhibiting cell proliferation and migration  <cit> .
it is noteworthy that we detected the stem cell marker, hoxb <dig>  as this finding supports a recent report that stem cells may dispersed along both sides of the crypt base  <cit>  and suggests that hoxb <dig> may be a biomarker of stem cells residing in the human ileum.
taken together, these data highlight a substantial presence of intestinal stem cells within the human ileal crypt, and suggest that ephb <dig>  hoxb <dig>  and sema3f may function in concert to regulate differentiation of epithelial progenitor cells and control the numbers and/or positioning of small intestinal paneth cells.
transcriptional programs in villus epithelial cells
epithelial cells isolated from the intestinal villi displayed increased transcription of a set of genes associated with epithelial cell differentiation , adhesion , motility , and cytoprotection  that was distinct from the set detected in crypt epithelial cells .
elevated mucin expression in the villus epithelial cell population was expected and supported by the large number of goblet cells that typically populate ileal villi .
in addition to the increased mucin, we detected enriched expression of mhc class ia, ic, and if molecules and fc fragment of igg binding protein , potentially indicative of the presence of intraepithelial lymphocytes within the epithelial lining.
these findings were consistent with the likelihood that several epithelial cell types known to reside in the villus region were isolated by lcmd.
transcriptional patterns of ileal epithelial biomarkers along the gi tract
to determine if the differentially expressed genes along the crypt-villus axis in the ileum are expressed at other regions of the gastrointestinal tract, we compared transcript levels of selected genes in the mucosa of the esophagus, duodenum, jejunum, ileum, and colon by rt-pcr .
villus epithelial cell biomarkers were also expressed at several locations along the gastrointestinal tract, yet, all appeared to be more highly transcribed in ileal tissues than at other sites.
eps8l <dig> and muc3b transcripts were detected in the ileum and colon, while aldolase-b was almost exclusively detected in the ileum.
interestingly, we found that paneth cell markers hd <dig> 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 ephrb <dig> were transcribed predominantly at other sites, including the colon  and esophagus .
discussion
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  <cit> , 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 a <dig> in mammalian small intestinal paneth cells have been documented  <cit> , 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 hd <dig> , apparently transcribed along the alimentary tract exclusively within the small bowel.
like hd <dig>  <cit> , reg3a is known as a downstream wnt/beta-catenin signaling target  <cit> .
the murine ortholog of reg3a , is expressed in paneth cells  <cit> .
while this localization is notable in light of studies demonstrating its antimicrobial properties  <cit> , reg3a has also been shown to stimulate liver regeneration and growth  <cit> .
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, rab <dig>  although little is yet known about rab <dig> 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  <cit> .
these investigations showed that rab <dig> 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 rab <dig> 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 .
our observation of considerable goblet cell numbers lining the villus epithelium in the ileal tissue samples utilized for lcmd  was reflected by increased mrna levels of mucin  <dig> and mucin 3b .
among the transport associated genes we found enriched in the villus epithelium were several members of the solute carrier family .
it is notable that this list included the oligopeptide transporter slc15a <dig> and the amino acid transporter slc7a <dig>  both recognized as villus specific biomarkers, and shown to be expressed at high levels in the mouse ileum  <cit> .
while much of the previous data on slc15a <dig> and slc7a <dig> 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 slc15a <dig> and slc7a <dig>  we detected enriched expression of the sodium/hydrogen exchanger slc9a <dig> and the sulfate transporter slc26a <dig> 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 .
among the enriched genes in the villus epithelium controlling growth and differentiation, reg <dig> was particularly interesting due to its role in activating the egf receptor  <cit> , and its classification, like paneth cell reg3a, within the regenerating  family of genes  <cit> .
thus, the large discrepancy between mrna levels of reg <dig> in the villus epithelium and reg3a in crypt epithelium highlights that differential expression of this gene family may serve as useful indicators  of differentiation in human ileal epithelial cell lineages.
CONCLUSIONS
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.

 epithelial stem cell progenitors, which reside in the intestinal crypt compartment, differentiate into either secretory or absorptive 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. 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. goblet cells perform cytoprotective, anti-inflammatory, and maintenance functions through synthesis and secretion of trefoil factors and mucins. 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. 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, and of wnt and notch signaling in the differentiation of secretory populations. several biomarkers believed to mediate differentiation of progenitor stem cells have also been identified. 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 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 to elucidate differential gene expression profiles of small intestinal villus and crypt epithelial cells, these cell populations were excised from unstained, sectioned formalin-fixed human ileal tissue samples by lcmd. the human ileal epithelial layer consists of multiple cell types that simultaneously differentiate from stem cell progenitors as they migrate toward their sites of function along the villus-crypt axis. villus epithelial cells were identified in unstained specimens based on topographical location; crypt epithelial cells were identified based on topographical location and appearance of granular cytoplasm characteristic of paneth cells. following rna extraction, linear amplification, labeling, and hybridization to dna microarrays, the genes for which the level of detected transcripts differed by at least 2-fold between the two cell pools were identified. these genes were then hierarchically clustered to identify common patterns of differential expression. genes that were expressed predominantly in either villus or crypt epithelia were then statistically evaluated to determine which pathways and processes were most represented. genes associated with protein biosynthesis, cellular stress and antimicrobial responses, phosphorylation, and cell cycle were enhanced in the crypt epithelial cells, while villus epithelial cells displayed increased expression of genes associated with lipid and carboxylic acid metabolism, transport, egf-like activity, and extracellular matrix functions. a list of the genes differentially transcribed in these categories, their fold differences in expression between crypt and villus epithelial cells, and the statistical confidence associated with those differences, is provided in additional file. the entire microarray data set is available for viewing at the gene expression omnibus database. as expected, crypt epithelial cells expressed dramatically higher levels of antimicrobial peptides characteristic of paneth cells, including alpha-defensin-, hd lysozyme, secretory phospholipase a and reg3a. race-pcr analysis confirmed that the alpha, and not the gamma, isoform of reg was expressed in the human ileal tissue, which contrasts with the nomenclature used in the mouse where reg3γ is expressed. trypsin, the proteolytic enzyme associated with alpha-defensin processing was also identified in the crypt. villus epithelial cells, in contrast, displayed markedly increased mrna levels of aldolase-b, apolipoprotein-a aminopeptidase-n, pep carboxykinase, and carboxylesterase- the identification of these previously established intestinal epithelial biomarkers provided evidence that our isolation of the desired epithelial lineages was successful, and validated the reproducibility of the methodology using formalin-fixed paraffin-embedded samples across multiple biological replicates. supportive evidence for microarray findings to provide further support that the transcriptional profiles detected by microarray analysis accurately represented the differences between villus and crypt epithelial cells, we evaluated expression of selected genes by immunohistochemistry. in agreement with transcriptome assessments and previous studies, we detected the antimicrobial peptide hd exclusively in paneth cells within crypt regions. aldolase-b, associated with carbohydrate metabolism in contrast, was detected primarily in epithelial cells lining the upper villus region within the same tissue sections. these data served to both substantiate the corresponding microarray findings at the protein level, and provide further support for our interpretations of the global transcriptome data for crypt and villus epithelial cell populations. to further test if the microarray analysis accurately represented the differences in mrna levels between villus and crypt epithelial cells, we evaluated expression of four genes by rt-pcr using the cdna pools obtained by lcmd. expression of hd and reg3a mrna was readily detected in the crypt epithelial cell cdna pool, but was not evident in the villus epithelial cell cdna under the same conditions. by contrast, aldolase b was detected in the villus epithelial cell cdna sample cycles sooner than in the crypt epithelial cell cdna sample, suggesting a difference in mrna levels of approximately × fold. as a control, β-actin was detected in both cdna pools at approximately the same levels. these data are consistent with the differential expression patterns of these genes detected by microarray hybridization analysis. transcriptional profiling of ileal crypt epithelium to elucidate potential mechanisms of paneth cell function, we evaluated candidate genes from the microarray data that were enriched in the crypt epithelial cell pool and believed to be associated with immune responses, cellular growth, differentiation, and motility. one striking example, reg3a was transcribed, on average, at > fold higher levels in crypt epithelial cells than measured in villus epithelial cells. reg3a has been shown previously to be expressed in intestinal paneth cells and has been shown to have both anti-inflammatory and antimicrobial properties. our findings indicate that reg3a is constitutively transcribed at substantial levels, similar to hd and trypsin in paneth cells of the human ileum. these data suggest that in the healthy human ileum, reg3a may function predominantly in regulating bacterial colonization. increased resistin-like beta transcription was also detected in crypt epithelial cells. 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. other genes transcribed preferentially in crypt epithelial cells included several involved in epithelial growth and positioning that may represent a distinct set of biomarkers specific to this cellular compartment. these included eph receptor b homeobox b and semaphorin 3f, implicated in previous studies to be involved in either the migration or specification of intestinal epithelial stem cell population. ephrin b and its receptor are believed to be regulated by wnt and expressed in crypt epithelia where they control the proper segregation of paneth cells from post-mitotic cells. homeobox b has been shown to regulate enteric development, while several investigations have highlighted the role of semaphorin 3f in inhibiting cell proliferation and migration. it is noteworthy that we detected the stem cell marker, hoxb as this finding supports a recent report that stem cells may dispersed along both sides of the crypt base and suggests that hoxb may be a biomarker of stem cells residing in the human ileum. taken together, these data highlight a substantial presence of intestinal stem cells within the human ileal crypt, and suggest that ephb hoxb and sema3f may function in concert to regulate differentiation of epithelial progenitor cells and control the numbers and/or positioning of small intestinal paneth cells. transcriptional programs in villus epithelial cells epithelial cells isolated from the intestinal villi displayed increased transcription of a set of genes associated with epithelial cell differentiation, adhesion, motility, and cytoprotection that was distinct from the set detected in crypt epithelial cells. elevated mucin expression in the villus epithelial cell population was expected and supported by the large number of goblet cells that typically populate ileal villi. in addition to the increased mucin, we detected enriched expression of mhc class ia, ic, and if molecules and fc fragment of igg binding protein, potentially indicative of the presence of intraepithelial lymphocytes within the epithelial lining. these findings were consistent with the likelihood that several epithelial cell types known to reside in the villus region were isolated by lcmd. transcriptional patterns of ileal epithelial biomarkers along the gi tract to determine if the differentially expressed genes along the crypt-villus axis in the ileum are expressed at other regions of the gastrointestinal tract, we compared transcript levels of selected genes in the mucosa of the esophagus, duodenum, jejunum, ileum, and colon by rt-pcr. villus epithelial cell biomarkers were also expressed at several locations along the gastrointestinal tract, yet, all appeared to be more highly transcribed in ileal tissues than at other sites. eps8l and muc3b transcripts were detected in the ileum and colon, while aldolase-b was almost exclusively detected in the ileum. interestingly, we found that paneth cell markers hd 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 ephrb were transcribed predominantly at other sites, including the colon and esophagus. discussion 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, 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 a in mammalian small intestinal paneth cells have been documented, 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 hd, apparently transcribed along the alimentary tract exclusively within the small bowel. like hd, reg3a is known as a downstream wnt/beta-catenin signaling target. the murine ortholog of reg3a, is expressed in paneth cells. while this localization is notable in light of studies demonstrating its antimicrobial properties, 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, rab although little is yet known about rab 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. these investigations showed that rab 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 rab 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. our observation of considerable goblet cell numbers lining the villus epithelium in the ileal tissue samples utilized for lcmd was reflected by increased mrna levels of mucin and mucin 3b. among the transport associated genes we found enriched in the villus epithelium were several members of the solute carrier family. it is notable that this list included the oligopeptide transporter slc15a and the amino acid transporter slc7a 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 slc15a and slc7a 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 slc15a and slc7a we detected enriched expression of the sodium/hydrogen exchanger slc9a and the sulfate transporter slc26a 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. among the enriched genes in the villus epithelium controlling growth and differentiation, reg was particularly interesting due to its role in activating the egf receptor, and its classification, like paneth cell reg3a, within the regenerating family of genes. thus, the large discrepancy between mrna levels of reg in the villus epithelium and reg3a in crypt epithelium highlights that differential expression of this gene family may serve as useful indicators 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. 
expression profiles in villus and crypt epithelium were determined by dna microarray, quantitative real-time pcr, and immunohistochemistry based methods. the spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. 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. 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. additionally, high baseline expression levels of innate antimicrobials, alpha defensin and regenerating islet-derived alpha, were detected exclusively within the small bowel crypt, most notably in the ileum in comparison to other sites along the gastrointestinal tract. 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.
 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. additionally, high baseline expression levels of innate antimicrobials, alpha defensin and regenerating islet-derived alpha, 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. 