- Email: [email protected]
Zeroing in on the “holy grail”
266
SELECTED SUMMARIES
GASTROENTEROLOGY Vol. 125, No. 1
is reached, at a new level of knowledge. Thankfully, it looks like we have now reached this period in the field of ICC research. Constipation is a common and heterogeneous disorder and can be divided into normal transit constipation, slow transit constipation (STC), and pelvic floor dysfunction based on transit and stool expulsion tests. By definition, STC is a somewhat more homogenous disease because delayed colonic transit is required for the diagnosis. Several studies have shown a decrease in the number of ICCs in STC requiring surgery. Whether the loss in ICCs is primary or secondary is not known; however, it is known that loss of ICCs results in abnormal motility (Gastroenterology 1998;114:724 –736). Therefore, even if the loss of ICCs is secondary, an understanding on how and why ICCs are lost is needed because they are ultimately required for the restoration of colonic motility and appear to be more plastic than nerves. Patients who come to surgery as a treatment of their STC have failed aggressive medical management and typically have used every available laxative without satisfactory relief. Unfortunately, it is unlikely that they can be further treated with laxatives to attempt to reverse an ICC defect. The question of whether colonic dilatation can be the cause of the observed loss of ICC is an interesting one. By custom, the diagnosis of STC is reserved for patients with slow transit without visible (on x-ray studies) dilatation. Colonic distention usually categorizes the disease process as colonic pseudo-obstruction. This is collaborated at surgery as the surgeon typically comments on a normal sized, albeit sometimes tortuous colon and at pathology when the pathologist reports a normal-appearing colon; this was the case in all patients reported in our recent studies (Gut 2002;51:496 –501, Gastroenterology 2000;118:14 –21). The question of dilatation resulting in loss of ICCs was raised by a study in mice, in which a subtotal obstruction was established in the ileum (J Physiol 2001;536:555–568). ICCs were lost, with a gradient of loss of ICCs up to 100 mm from the clip. Whether the same occurs in the human small intestine or colon in response to marked dilatation is not known. Patients with intestinal pseudo-obstruction have been reported to have loss of ICCs, but other reports also show that some patients with pseudo-obstruction have a normal complement of ICCs, suggesting that loss of ICCs is not an automatic sequelae of intestinal dilatation in humans. A recent study on patients with pseudo-obstruction used as controls resected specimens from patients with either Crohn’s disease or colorectal cancer, including specimens with preobstructive dilatation (Am J Surg Pathol 2003;27:228 –235). No loss of ICCs was noted in the controls with preobstructive dilatation. It may be that differences are present between the human and murine response to dilatation or that the level of dilatation achieved in the murine small intestine is not easily achieved in the human. Severe STC is a disease with a high morbidity and with a significant impact on daily quality of life. We completely concur that much work still needs to be done to determine definitely the pathophysiology of the disease and hopefully to impact on its diagnosis and management. Disorders in ICC are likely to play a role in the pathophysiology of STC in a subset of patients but are probably equally unlikely to be the only cause. GIANRICO FARRUGIA, M.D.
ZEROING IN ON THE “HOLY GRAIL” Mills JC, Andersson N, Hong CV, Stappenbeck TS, Gordon JI (Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri).
Molecular characterization of mouse gastric epithelial progenitor cells. Proc Natl Acad Sci U S A 2002;99:14819 –14824. Stem cells are the focus of an intense research effort because of their ability both to self-renew and to differentiate into a variety of tissues. In previous work by the Gordon laboratory (Proc Natl Acad Sci U S A 2000;97:12601–12606), characterization of intestinal stem cells has been referred to as the “Holy Grail” for gastrointestinal researchers. In this current article, they have taken a major step in providing that characterization. This depiction is in the form of gene expression profiles produced from DNA microarray gene chips using intact stomach tissue enriched for gastric epithelial progenitor (GEP) cells compared with the normal adult. Specifically, 25% of epithelial cells in the gastric units of tox176 mice are GEPs (J Biol Chem 1996;271:3671–3676); 18-day embryonic mice (E18) have ⬎90% (Am J Physiol 1997;272:G1209 –G1220), whereas the normal mouse has ⬍3% (Mol Cell 1997;3:263– 274). A GEP dataset of 147 genes with increased expression in both tox176 and E18 mice over normal adult stomach in duplicate runs was generated. The GEP dataset was validated using 4 different methods. Quantitative real time PCR (qRT-PCR) for 7 selected genes in the GEP dataset exhibited increased messenger RNA (mRNA) levels of 2–9-fold in tox176 and E18 versus normal stomach. Immunohistochemical comparisons (light and electron microscopic methods) between tox176 and normal gastric tissue for 5 proteins from the dataset where found to be higher in the GEP-enriched material. Navigated laser capture microscopy (n-LCM) was used to isolate cells from gastric unit compartments (pit, isthmus, base) with multiple stains used as a guide to discriminate cell types. The RNA extracted from the isolated cells was evaluated by qRT-PCR for 4 selected genes, all of which exhibited increased levels of expression over normal mouse RNA. Comparison of the GEP dataset with hematopoietic stem cell (HSC) datasets, parietal cells and zymogenic cells (two differentiated descendents of the GEPs), was the final validation step. The authors made their comparisons with other stem cell and non-stem cell gene profiles by giving each gene a functional assignment based on the Gene Ontology (GO) consortium classification scheme (www.genoeontology.org), which automatically classify the genes in the datasets. Fractional representation defined as the number of genes with a given GO classification in a list relative to the total number of genes in that list was used to make the comparisons. Results showed that the GEP dataset was functionally similar to the dataset of the HSCs but much different than datasets from either of the descendent cells. The investigators were able to characterize further many of the GEP dataset genes into 3 functional categories: components of the growth factor pathway, regulators of protein turnover, and mRNA processing and cytoplasmic localization. Based on the analysis of the GEP dataset, it was proposed that insulin-like growth factor (IGF) genes might play a significant
July 2003
role in epithelial stem cell biology. Furthermore, hypothesizing that processing and cytoplasmic localization may have different roles in different areas of the gastrointestinal tract, the investigators studied 3 selected genes with high gastric GEP cell expression. The genes were evaluated in both progenitor and differentiated locations for the small bowel and colon using nLCM/qRT-PCR. They found that 2 showed increases in small bowel and colon while a third was undetectable. Comment. This work provides one of the first indications of the molecular nature of intestinal epithelial stem cells. Using DNA microarray technology, the authors determined a unique mRNA expression pattern in enriched populations of gastric epithelial progenitor cells. Lacking definitive markers, (J Clin Invest 2000;105: 1493–1499) the extensive validation process undertaken in this work was necessary to confirm the identity of the progenitor cells. Likewise, grouping genes into functional categories served to identify genes that may be expected to have increased expression in stem cells. More interesting is the proposal that IGFs and related genes are key effectors of epithelial stem cell biology and census. These IGFs may help the GEP cells maintain their niche location within the intestinal epithelium although other factors have also been proposed (Nature 2001;414:98 –104). Because of the asymmetrical division of epithelial stem cells, cytoplasmic localization genes that define polarity and development of axis formation are important indicators of stemness (BioEssays 2002;24:91–98). For a very limited number of these localization genes, the authors showed differences between the gastric unit and small intestine/colon epithelium reinforcing their overall claim. All of this work strongly supports the GEP dataset as a valid first approximation of the epithelial stem cell molecular signature. Currently, one of the best methods to identify stem cells is using mRNA profiles. Common patterns of gene expression are seen between divergent sources of progenitor cells (Science 2002;298:601– 604). Thus far, no uniquely stem cell genes have been identified in any of the systems studied. Perhaps the overall pattern of gene expression is what is important in defining stem cells rather then the existence of novel genes. Because of the variability and sensitivity issues inherent in DNA microarray experiments, further characterization will be necessary to define completely epithelial “stemness” at the mRNA level. A broader problem is that mRNA expression levels may not represent the levels of protein expression. In one report, mRNA and protein levels were found to correlate only about 20% of the time (Mol Cell Proteomics 2002;1:304 –313). Also, nothing can be directly learned of posttranslation protein modification or other aspects of the actual workings of the protein in the stem cell by knowing the mRNA expression level alone. To truly understand the biology of stem cells, work at the protein level will be vital. Combining n-LCM, a method developed by the Gordon laboratory (Proc Natl Acad Sci U S A 2000;97:12601–12606), with qRT-PCR was very effective in showing highly enriched GEP genes in the isthmus area of the gastric unit, where the stem cells are expected to reside. This emphasizes the special value of LCM in the study of very selective cell populations within complex tissue of many differing types. No other method offers the precision of extraction with minimal disruption of material that LCM affords. Using n-LCM and genes identified as locally unique, direct isolation of intestinal epithelial stem cells should be possible. Linear amplification of RNA isolated from n-LCM can be used to probe complementary DNA (cDNA) microarrays producing a more extensive and complete determination of the epithelial stem cell molecular signature. The devel-
SELECTED SUMMARIES
267
oping technology of combining LCM material with mass spectrometry is beginning to provide a method to analyze the proteins of these selected cells. LCM will continue to be a vital tool in the investigation of intestinal epithelial stem cells. This study is a significant contribution towards the molecular characterization of intestinal epithelial stem cells. The combination of cDNA microarray, n-LCM, and qRT-PCR methods was very efficiently used to identify and validate a stem cell molecular signature and represents the best technical approach for the problem. Most importantly, the mRNA profile outlined may provide the means for more detailed analysis of progenitor cells. This will consequently advance our understanding of the biology of the gut as well as the nature of many intestinal diseases. CHRISTOPHER R. ERWIN, PH.D. MARCUS JARBOE, M.D. BRAD W. WARNER, M.D.
Reply. As many of us seek to identify and characterize the regulatory networks that control epithelial renewal in the mammalian gut, laser-capture microdissection (LCM) is a valuable tool for extracting gut epithelial lineage progenitors directly from their niche. We have recently used LCM to retrieve small intestinal epithelial progenitors (SiEPs) from germ-free transgenic mice with a genetically engineered ablation of their Paneth cells (a key component of the gut’s innate immune system). The problem with retrieving SiEPs from cryosections is that they are normally rare and interspersed between numerous differentiated Paneth cells. Paneth cell ablation results in physical consolidation of SiEPs in a discrete region of the small intestine’s crypts of Lieberku¨ hn—the crypt base. This consolidation into common space makes them succulent targets for an LCM harvest. Subsequent generation of a SiEP gene expression profile, using DNA microarrays, disclosed notable similarities to gastric epithelial lineage progenitors, and to neural and hematopoietic stem cells (Proc Natl Acad Sci U S A 2003;100:1004 –1009). These findings are tantalizing. They evoke a feeling (and among the more faithful, a belief) that we are seeing the tip of an iceberg, and that beneath the surface lurks an elaborate ensemble of molecules and regulatory circuits shared by many types of progenitors, even those that reside in what superficially appear to be quite different tissue microenvironments (Science 2002; 298:597– 600). Generating and then comparing hundreds or thousands of genes present in a DNA microarray-based profile of one type of progenitor, with comparably sized datasets obtained from another type of progenitor, can be daunting. Identifying and understanding the significance of shared as well as seemingly disparate features is intimidating, but presents an inspiring challenge. Obtaining answers requires ongoing development of new computational approaches for categorizing and comparing these large datasets. One avenue that we are following, gene ontology (GO)-based bioinformatics tools that allow more objective and automated functional categorization of these profiles (Mills J, Carmichael L, Gordon J, unpublished observations), is just a small part of a community-wide effort to provide a common language for comparing datasets that will soon emerge from multicenter stem cell genome anatomy projects (e.g., http://www.scgap. org). For the present day Galahads, Gawains, and Arthurs pursuing the quest(ion) of what defines and determines “stemness,” such metaanalyses should provide inspiring thoughts about the nature and meaning of this long sought after chalice. JASON C. MILLS, M.D., Ph.D. THADDEUS S. STAPPENBECK, M.D., Ph.D. JEFFREY I. GORDON, M.D.
SELECTED SUMMARIES
GASTROENTEROLOGY Vol. 125, No. 1
is reached, at a new level of knowledge. Thankfully, it looks like we have now reached this period in the field of ICC research. Constipation is a common and heterogeneous disorder and can be divided into normal transit constipation, slow transit constipation (STC), and pelvic floor dysfunction based on transit and stool expulsion tests. By definition, STC is a somewhat more homogenous disease because delayed colonic transit is required for the diagnosis. Several studies have shown a decrease in the number of ICCs in STC requiring surgery. Whether the loss in ICCs is primary or secondary is not known; however, it is known that loss of ICCs results in abnormal motility (Gastroenterology 1998;114:724 –736). Therefore, even if the loss of ICCs is secondary, an understanding on how and why ICCs are lost is needed because they are ultimately required for the restoration of colonic motility and appear to be more plastic than nerves. Patients who come to surgery as a treatment of their STC have failed aggressive medical management and typically have used every available laxative without satisfactory relief. Unfortunately, it is unlikely that they can be further treated with laxatives to attempt to reverse an ICC defect. The question of whether colonic dilatation can be the cause of the observed loss of ICC is an interesting one. By custom, the diagnosis of STC is reserved for patients with slow transit without visible (on x-ray studies) dilatation. Colonic distention usually categorizes the disease process as colonic pseudo-obstruction. This is collaborated at surgery as the surgeon typically comments on a normal sized, albeit sometimes tortuous colon and at pathology when the pathologist reports a normal-appearing colon; this was the case in all patients reported in our recent studies (Gut 2002;51:496 –501, Gastroenterology 2000;118:14 –21). The question of dilatation resulting in loss of ICCs was raised by a study in mice, in which a subtotal obstruction was established in the ileum (J Physiol 2001;536:555–568). ICCs were lost, with a gradient of loss of ICCs up to 100 mm from the clip. Whether the same occurs in the human small intestine or colon in response to marked dilatation is not known. Patients with intestinal pseudo-obstruction have been reported to have loss of ICCs, but other reports also show that some patients with pseudo-obstruction have a normal complement of ICCs, suggesting that loss of ICCs is not an automatic sequelae of intestinal dilatation in humans. A recent study on patients with pseudo-obstruction used as controls resected specimens from patients with either Crohn’s disease or colorectal cancer, including specimens with preobstructive dilatation (Am J Surg Pathol 2003;27:228 –235). No loss of ICCs was noted in the controls with preobstructive dilatation. It may be that differences are present between the human and murine response to dilatation or that the level of dilatation achieved in the murine small intestine is not easily achieved in the human. Severe STC is a disease with a high morbidity and with a significant impact on daily quality of life. We completely concur that much work still needs to be done to determine definitely the pathophysiology of the disease and hopefully to impact on its diagnosis and management. Disorders in ICC are likely to play a role in the pathophysiology of STC in a subset of patients but are probably equally unlikely to be the only cause. GIANRICO FARRUGIA, M.D.
ZEROING IN ON THE “HOLY GRAIL” Mills JC, Andersson N, Hong CV, Stappenbeck TS, Gordon JI (Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri).
Molecular characterization of mouse gastric epithelial progenitor cells. Proc Natl Acad Sci U S A 2002;99:14819 –14824. Stem cells are the focus of an intense research effort because of their ability both to self-renew and to differentiate into a variety of tissues. In previous work by the Gordon laboratory (Proc Natl Acad Sci U S A 2000;97:12601–12606), characterization of intestinal stem cells has been referred to as the “Holy Grail” for gastrointestinal researchers. In this current article, they have taken a major step in providing that characterization. This depiction is in the form of gene expression profiles produced from DNA microarray gene chips using intact stomach tissue enriched for gastric epithelial progenitor (GEP) cells compared with the normal adult. Specifically, 25% of epithelial cells in the gastric units of tox176 mice are GEPs (J Biol Chem 1996;271:3671–3676); 18-day embryonic mice (E18) have ⬎90% (Am J Physiol 1997;272:G1209 –G1220), whereas the normal mouse has ⬍3% (Mol Cell 1997;3:263– 274). A GEP dataset of 147 genes with increased expression in both tox176 and E18 mice over normal adult stomach in duplicate runs was generated. The GEP dataset was validated using 4 different methods. Quantitative real time PCR (qRT-PCR) for 7 selected genes in the GEP dataset exhibited increased messenger RNA (mRNA) levels of 2–9-fold in tox176 and E18 versus normal stomach. Immunohistochemical comparisons (light and electron microscopic methods) between tox176 and normal gastric tissue for 5 proteins from the dataset where found to be higher in the GEP-enriched material. Navigated laser capture microscopy (n-LCM) was used to isolate cells from gastric unit compartments (pit, isthmus, base) with multiple stains used as a guide to discriminate cell types. The RNA extracted from the isolated cells was evaluated by qRT-PCR for 4 selected genes, all of which exhibited increased levels of expression over normal mouse RNA. Comparison of the GEP dataset with hematopoietic stem cell (HSC) datasets, parietal cells and zymogenic cells (two differentiated descendents of the GEPs), was the final validation step. The authors made their comparisons with other stem cell and non-stem cell gene profiles by giving each gene a functional assignment based on the Gene Ontology (GO) consortium classification scheme (www.genoeontology.org), which automatically classify the genes in the datasets. Fractional representation defined as the number of genes with a given GO classification in a list relative to the total number of genes in that list was used to make the comparisons. Results showed that the GEP dataset was functionally similar to the dataset of the HSCs but much different than datasets from either of the descendent cells. The investigators were able to characterize further many of the GEP dataset genes into 3 functional categories: components of the growth factor pathway, regulators of protein turnover, and mRNA processing and cytoplasmic localization. Based on the analysis of the GEP dataset, it was proposed that insulin-like growth factor (IGF) genes might play a significant
July 2003
role in epithelial stem cell biology. Furthermore, hypothesizing that processing and cytoplasmic localization may have different roles in different areas of the gastrointestinal tract, the investigators studied 3 selected genes with high gastric GEP cell expression. The genes were evaluated in both progenitor and differentiated locations for the small bowel and colon using nLCM/qRT-PCR. They found that 2 showed increases in small bowel and colon while a third was undetectable. Comment. This work provides one of the first indications of the molecular nature of intestinal epithelial stem cells. Using DNA microarray technology, the authors determined a unique mRNA expression pattern in enriched populations of gastric epithelial progenitor cells. Lacking definitive markers, (J Clin Invest 2000;105: 1493–1499) the extensive validation process undertaken in this work was necessary to confirm the identity of the progenitor cells. Likewise, grouping genes into functional categories served to identify genes that may be expected to have increased expression in stem cells. More interesting is the proposal that IGFs and related genes are key effectors of epithelial stem cell biology and census. These IGFs may help the GEP cells maintain their niche location within the intestinal epithelium although other factors have also been proposed (Nature 2001;414:98 –104). Because of the asymmetrical division of epithelial stem cells, cytoplasmic localization genes that define polarity and development of axis formation are important indicators of stemness (BioEssays 2002;24:91–98). For a very limited number of these localization genes, the authors showed differences between the gastric unit and small intestine/colon epithelium reinforcing their overall claim. All of this work strongly supports the GEP dataset as a valid first approximation of the epithelial stem cell molecular signature. Currently, one of the best methods to identify stem cells is using mRNA profiles. Common patterns of gene expression are seen between divergent sources of progenitor cells (Science 2002;298:601– 604). Thus far, no uniquely stem cell genes have been identified in any of the systems studied. Perhaps the overall pattern of gene expression is what is important in defining stem cells rather then the existence of novel genes. Because of the variability and sensitivity issues inherent in DNA microarray experiments, further characterization will be necessary to define completely epithelial “stemness” at the mRNA level. A broader problem is that mRNA expression levels may not represent the levels of protein expression. In one report, mRNA and protein levels were found to correlate only about 20% of the time (Mol Cell Proteomics 2002;1:304 –313). Also, nothing can be directly learned of posttranslation protein modification or other aspects of the actual workings of the protein in the stem cell by knowing the mRNA expression level alone. To truly understand the biology of stem cells, work at the protein level will be vital. Combining n-LCM, a method developed by the Gordon laboratory (Proc Natl Acad Sci U S A 2000;97:12601–12606), with qRT-PCR was very effective in showing highly enriched GEP genes in the isthmus area of the gastric unit, where the stem cells are expected to reside. This emphasizes the special value of LCM in the study of very selective cell populations within complex tissue of many differing types. No other method offers the precision of extraction with minimal disruption of material that LCM affords. Using n-LCM and genes identified as locally unique, direct isolation of intestinal epithelial stem cells should be possible. Linear amplification of RNA isolated from n-LCM can be used to probe complementary DNA (cDNA) microarrays producing a more extensive and complete determination of the epithelial stem cell molecular signature. The devel-
SELECTED SUMMARIES
267
oping technology of combining LCM material with mass spectrometry is beginning to provide a method to analyze the proteins of these selected cells. LCM will continue to be a vital tool in the investigation of intestinal epithelial stem cells. This study is a significant contribution towards the molecular characterization of intestinal epithelial stem cells. The combination of cDNA microarray, n-LCM, and qRT-PCR methods was very efficiently used to identify and validate a stem cell molecular signature and represents the best technical approach for the problem. Most importantly, the mRNA profile outlined may provide the means for more detailed analysis of progenitor cells. This will consequently advance our understanding of the biology of the gut as well as the nature of many intestinal diseases. CHRISTOPHER R. ERWIN, PH.D. MARCUS JARBOE, M.D. BRAD W. WARNER, M.D.
Reply. As many of us seek to identify and characterize the regulatory networks that control epithelial renewal in the mammalian gut, laser-capture microdissection (LCM) is a valuable tool for extracting gut epithelial lineage progenitors directly from their niche. We have recently used LCM to retrieve small intestinal epithelial progenitors (SiEPs) from germ-free transgenic mice with a genetically engineered ablation of their Paneth cells (a key component of the gut’s innate immune system). The problem with retrieving SiEPs from cryosections is that they are normally rare and interspersed between numerous differentiated Paneth cells. Paneth cell ablation results in physical consolidation of SiEPs in a discrete region of the small intestine’s crypts of Lieberku¨ hn—the crypt base. This consolidation into common space makes them succulent targets for an LCM harvest. Subsequent generation of a SiEP gene expression profile, using DNA microarrays, disclosed notable similarities to gastric epithelial lineage progenitors, and to neural and hematopoietic stem cells (Proc Natl Acad Sci U S A 2003;100:1004 –1009). These findings are tantalizing. They evoke a feeling (and among the more faithful, a belief) that we are seeing the tip of an iceberg, and that beneath the surface lurks an elaborate ensemble of molecules and regulatory circuits shared by many types of progenitors, even those that reside in what superficially appear to be quite different tissue microenvironments (Science 2002; 298:597– 600). Generating and then comparing hundreds or thousands of genes present in a DNA microarray-based profile of one type of progenitor, with comparably sized datasets obtained from another type of progenitor, can be daunting. Identifying and understanding the significance of shared as well as seemingly disparate features is intimidating, but presents an inspiring challenge. Obtaining answers requires ongoing development of new computational approaches for categorizing and comparing these large datasets. One avenue that we are following, gene ontology (GO)-based bioinformatics tools that allow more objective and automated functional categorization of these profiles (Mills J, Carmichael L, Gordon J, unpublished observations), is just a small part of a community-wide effort to provide a common language for comparing datasets that will soon emerge from multicenter stem cell genome anatomy projects (e.g., http://www.scgap. org). For the present day Galahads, Gawains, and Arthurs pursuing the quest(ion) of what defines and determines “stemness,” such metaanalyses should provide inspiring thoughts about the nature and meaning of this long sought after chalice. JASON C. MILLS, M.D., Ph.D. THADDEUS S. STAPPENBECK, M.D., Ph.D. JEFFREY I. GORDON, M.D.