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Altered Stem Cell Dynamics in Human Colon Adenoma Crypts Allow Rapid Expansion and Fixation of Mutations During Clonal Expansion
AGA Abstracts
cells in human colon by using methylation patterns. Proc Natl Acad Sci USA 98(19):1083910844. 2) Graham TA et al. Inferring the expansion of stem cell clones in the human colon by using methylation patterns. Submitted to Gastroenterology (under review).
Figure 1: Classification rules using a step-up approach 52 The Effect of Early Aggressive Fluid Resuscitation on Morbidity and Mortality in Acute Pancreatitis Matthew G. Warndorf, Jane T. Kurtzman, Michael J. Bartel, Mougnyan Cox, Todd A. Mackenzie, Paul Burchard, Stuart R. Gordon, Timothy B. Gardner BACKGROUND & AIM: Early aggressive fluid resuscitation is a beneficial treatment for acute pancreatitis. However, the degree of this benefit is not known. We aimed to determine the effect of early aggressive fluid resuscitation on important outcomes such as SIRS, organ failure, and mortality in patients admitted with acute pancreatitis. METHODS: All nontransferred patients admitted to our medical center from 1985-2009 with a primary diagnosis of acute pancreatitis were identified and data extraction performed retrospectively. Early aggressive resuscitation was defined as receiving ≥ 1/3 of the total 72 hour fluid volume within the first 24 hours of presentation. Late resuscitation was defined as receiving ≤ 1/ 3 of the total 72 hour fluid volume within the first 24 hours of presentation. The primary study outcomes were the effect of fluid resuscitation on SIRS, organ failure, and mortality. Data were analyzed via standard chi-square analysis and student's t-tests, respectively and multivariate regression performed for confounders of age, comorbidity and gender. RESULTS: 434 patients were included in the analysis; 340 were categorized in the early resuscitation group and 94 in the late resuscitation group. Patients in the early resuscitation group received more IV fluids in the first 24 hours (3,493 ml vs. 2,402 ml, p < 0.001) but less total fluids in the first 72 hours (7,600 ml vs. 9,514 ml, p = 0.001) compared with the late resuscitation group. Early fluid resuscitation led to decreased SIRS at 24 (15% vs. 32%, p = 0.001), 48 (14% vs. 33%, p = 0.001), and 72 (10% vs. 23%, p = 0.01) hours respectively (p < 0.01) as well as decreased organ failure at 72 hours (5% vs. 10%, p < 0.05), a lower rate of ICU admission (6% vs. 17%, p < 0.001) and a shortened length of stay (8 vs. 11 days, p = 0.01) . Although not statistically significant, organ failure at 24 and 48 hours was also improved in the early resuscitation group. There was no significant difference in mortality between the two groups. Subgroup analysis demonstrated these benefits to be more pronounced in patients with interstitial rather than severe pancreatitis at admission. CONCLUSIONS: In patients with acute pancreatitis, early aggressive fluid resuscitation within the first twentyfour hours reduces the development of systemic inflammatory response syndrome (SIRS), organ failure, the need for ICU admission, and overall hospital length of stay in patients presenting with interstitial disease. In patients presenting with severe acute pancreatitis the benefit of early aggressive fluid resuscitation was not seen.
54 Single Cell Gene Expression Profiling of Adult Murine Colon Provides Novel Insight Into Both the Origin of Stem Cells and Their Supportive Cell Type Michael E. Rothenberg, Tomer Kalisky, Piero D. Dalerba, Ysbrand Nusse, Stephen Quake, Michael F. Clarke
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Intestinal stem cell markers Lgr5 and Bmi1 have been identified by In-Vivo lineage tracing experiments; however, it remains unclear whether these markers identify distinct or overlapping cell types. Furthermore, while a recent study showed that Paneth cells contribute significantly to the small intestinal Lgr5 stem cell niche, the existence of a similar cell type in the colon has not yet been firmly established. To address these questions, we have undertaken a prospective high-resolution characterization of adult murine colon using a combination of classical and novel techniques: flow cytometry with widely-available cell surface markers on live dissociated colonocytes, in-vitro culture of sorted cells into colonic “organoids,” and multiplexed qRT-PCR based single-cell gene expression analysis. We can sort two distinct colonic crypt-base subpopulations that both express the self-renewal gene telomerase and can generate organoids in-vitro, but only one of which contains Lgr5-high expressing cells. In agreement with this, single-cell gene expression analysis shows that Lgr5-high cells exhibit a transcriptional signature that is different from the Bmi1-high cells. Because this analysis represents a snapshot of gene expression, colon stem cells might exist in two different transcriptional states; alternatively, it remains possible that two different colon stem cell populations exist. Furthermore, our analysis demonstrates the existence at the crypt base of new subtypes of secretory Muc2+ (goblet) cells: A Krt20-positive cell and a Krt20-low/negative cell that elaborates several secreted factors implicated in crypt homeostasis and stem cell regulation (e.g., Wnt-activators, EGF, Indian Hedgehog, and Notch-pathway ligands Dll1 and Dll4). Similar analyses on human colon crypt base cells shows that the same cell types exist in humans and suggest that stem cell regulatory mechanisms are well conserved between mice and humans. Conclusions: -Lgr5-high and Bmi1-high cells exhibit mostly distinct transcriptional signatures in the adult mouse colon -A well-conserved goblet (Muc2+) cell that elaborates factors implicated in stem cell regulation (EGF, Notch activators, Wnt activators, and Hedgehog ligand) can be found at the crypt base, representing a possible source of niche-factors for colonic stem cells Funding Sources: Dr. Rothenberg is supported by a UCB IBD Working Group 2010 GI Fellows Research Award and a California Institute for Regenerative Medicine (CIRM) Scholar Training Grant TG2-01159
Altered Stem Cell Dynamics in Human Colon Adenoma Crypts Allow Rapid Expansion and Fixation of Mutations During Clonal Expansion Adam Humphries, Trevor A. Graham, Anna M. Nicholson, Stuart A. McDonald, Nicholas A. Wright Introduction: Methylation patterns at CpG islands within non-expressed genes are surrogate markers of cell ancestry and dynamics in normal human colon crypts, and have been used to provide evidence that crypts contain multiple stem cells within a niche1. Over time, a single stem cell and its progeny will come to occupy the whole niche - niche succession, and subsequently the crypt - monoclonal conversion. Crypts divide by a process of crypt fission, whereby a parent crypt produces two, clonally related daughter crypts, and this is the predominant method by which crypts expand in early tumorigenesis. By analysing methylation patterns of normal human colon crypts, known to be related by their sharing clonal point mutations in mitochondrial DNA (mtDNA), we previously found that the rate of niche succession is slow (years), and crypt fission is infrequent, occurring approximately once every 25 years2. There is a need to evaluate these dynamics in adenomas to understand how quickly they grow; we hypothesise that both crypt fission and niche succession occur at a faster rate in adenoma tissue enabling accelerated clonal expansion. Methods: Fresh frozen human adenomas were obtained and clonal patches of adenomatous crypts identified by combining enzyme histochemistry and laser capture microdissection with the use of PCR sequencing to demonstrate shared clonal point mutations in mtDNA. Using DNA from the same crypt, methylation patterns at CpG islands of 3 non-expressed genes were determined using clonal bisulphite sequencing. Statistical analysis was then performed to compare crypt methylation patterns. Results: In contrast to normal human colon tissue, multiple, large clonal patches of crypts that shared common point mutations in their mtDNA were identified within human adenomas; these recently related adenomatous crypts had very similar, conserved methylation patterns compared to unrelated adenomatous crypts (p=8.08 x10-12). Conclusions: This study demonstrates that increased rates of niche succession, monoclonal conversion and crypt fission enable accelerated clonal expansion of mutated crypts in human adenomas, suggesting that these up-regulated processes allow rapid fixation and spread of successive oncogenic mutations during early tumorigenesis; further data collection will allow the time course of these events to be estimated. Combining the techniques used here with clonal oncogenic changes may reveal the selective growth advantage of specific mutations within adenomas. References: 1) Yatabe Y, Tavaré S, & Shibata D (2001). Investigating stem
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AGA Abstracts
cells in human colon by using methylation patterns. Proc Natl Acad Sci USA 98(19):1083910844. 2) Graham TA et al. Inferring the expansion of stem cell clones in the human colon by using methylation patterns. Submitted to Gastroenterology (under review).
Figure 1: Classification rules using a step-up approach 52 The Effect of Early Aggressive Fluid Resuscitation on Morbidity and Mortality in Acute Pancreatitis Matthew G. Warndorf, Jane T. Kurtzman, Michael J. Bartel, Mougnyan Cox, Todd A. Mackenzie, Paul Burchard, Stuart R. Gordon, Timothy B. Gardner BACKGROUND & AIM: Early aggressive fluid resuscitation is a beneficial treatment for acute pancreatitis. However, the degree of this benefit is not known. We aimed to determine the effect of early aggressive fluid resuscitation on important outcomes such as SIRS, organ failure, and mortality in patients admitted with acute pancreatitis. METHODS: All nontransferred patients admitted to our medical center from 1985-2009 with a primary diagnosis of acute pancreatitis were identified and data extraction performed retrospectively. Early aggressive resuscitation was defined as receiving ≥ 1/3 of the total 72 hour fluid volume within the first 24 hours of presentation. Late resuscitation was defined as receiving ≤ 1/ 3 of the total 72 hour fluid volume within the first 24 hours of presentation. The primary study outcomes were the effect of fluid resuscitation on SIRS, organ failure, and mortality. Data were analyzed via standard chi-square analysis and student's t-tests, respectively and multivariate regression performed for confounders of age, comorbidity and gender. RESULTS: 434 patients were included in the analysis; 340 were categorized in the early resuscitation group and 94 in the late resuscitation group. Patients in the early resuscitation group received more IV fluids in the first 24 hours (3,493 ml vs. 2,402 ml, p < 0.001) but less total fluids in the first 72 hours (7,600 ml vs. 9,514 ml, p = 0.001) compared with the late resuscitation group. Early fluid resuscitation led to decreased SIRS at 24 (15% vs. 32%, p = 0.001), 48 (14% vs. 33%, p = 0.001), and 72 (10% vs. 23%, p = 0.01) hours respectively (p < 0.01) as well as decreased organ failure at 72 hours (5% vs. 10%, p < 0.05), a lower rate of ICU admission (6% vs. 17%, p < 0.001) and a shortened length of stay (8 vs. 11 days, p = 0.01) . Although not statistically significant, organ failure at 24 and 48 hours was also improved in the early resuscitation group. There was no significant difference in mortality between the two groups. Subgroup analysis demonstrated these benefits to be more pronounced in patients with interstitial rather than severe pancreatitis at admission. CONCLUSIONS: In patients with acute pancreatitis, early aggressive fluid resuscitation within the first twentyfour hours reduces the development of systemic inflammatory response syndrome (SIRS), organ failure, the need for ICU admission, and overall hospital length of stay in patients presenting with interstitial disease. In patients presenting with severe acute pancreatitis the benefit of early aggressive fluid resuscitation was not seen.
54 Single Cell Gene Expression Profiling of Adult Murine Colon Provides Novel Insight Into Both the Origin of Stem Cells and Their Supportive Cell Type Michael E. Rothenberg, Tomer Kalisky, Piero D. Dalerba, Ysbrand Nusse, Stephen Quake, Michael F. Clarke
53
Intestinal stem cell markers Lgr5 and Bmi1 have been identified by In-Vivo lineage tracing experiments; however, it remains unclear whether these markers identify distinct or overlapping cell types. Furthermore, while a recent study showed that Paneth cells contribute significantly to the small intestinal Lgr5 stem cell niche, the existence of a similar cell type in the colon has not yet been firmly established. To address these questions, we have undertaken a prospective high-resolution characterization of adult murine colon using a combination of classical and novel techniques: flow cytometry with widely-available cell surface markers on live dissociated colonocytes, in-vitro culture of sorted cells into colonic “organoids,” and multiplexed qRT-PCR based single-cell gene expression analysis. We can sort two distinct colonic crypt-base subpopulations that both express the self-renewal gene telomerase and can generate organoids in-vitro, but only one of which contains Lgr5-high expressing cells. In agreement with this, single-cell gene expression analysis shows that Lgr5-high cells exhibit a transcriptional signature that is different from the Bmi1-high cells. Because this analysis represents a snapshot of gene expression, colon stem cells might exist in two different transcriptional states; alternatively, it remains possible that two different colon stem cell populations exist. Furthermore, our analysis demonstrates the existence at the crypt base of new subtypes of secretory Muc2+ (goblet) cells: A Krt20-positive cell and a Krt20-low/negative cell that elaborates several secreted factors implicated in crypt homeostasis and stem cell regulation (e.g., Wnt-activators, EGF, Indian Hedgehog, and Notch-pathway ligands Dll1 and Dll4). Similar analyses on human colon crypt base cells shows that the same cell types exist in humans and suggest that stem cell regulatory mechanisms are well conserved between mice and humans. Conclusions: -Lgr5-high and Bmi1-high cells exhibit mostly distinct transcriptional signatures in the adult mouse colon -A well-conserved goblet (Muc2+) cell that elaborates factors implicated in stem cell regulation (EGF, Notch activators, Wnt activators, and Hedgehog ligand) can be found at the crypt base, representing a possible source of niche-factors for colonic stem cells Funding Sources: Dr. Rothenberg is supported by a UCB IBD Working Group 2010 GI Fellows Research Award and a California Institute for Regenerative Medicine (CIRM) Scholar Training Grant TG2-01159
Altered Stem Cell Dynamics in Human Colon Adenoma Crypts Allow Rapid Expansion and Fixation of Mutations During Clonal Expansion Adam Humphries, Trevor A. Graham, Anna M. Nicholson, Stuart A. McDonald, Nicholas A. Wright Introduction: Methylation patterns at CpG islands within non-expressed genes are surrogate markers of cell ancestry and dynamics in normal human colon crypts, and have been used to provide evidence that crypts contain multiple stem cells within a niche1. Over time, a single stem cell and its progeny will come to occupy the whole niche - niche succession, and subsequently the crypt - monoclonal conversion. Crypts divide by a process of crypt fission, whereby a parent crypt produces two, clonally related daughter crypts, and this is the predominant method by which crypts expand in early tumorigenesis. By analysing methylation patterns of normal human colon crypts, known to be related by their sharing clonal point mutations in mitochondrial DNA (mtDNA), we previously found that the rate of niche succession is slow (years), and crypt fission is infrequent, occurring approximately once every 25 years2. There is a need to evaluate these dynamics in adenomas to understand how quickly they grow; we hypothesise that both crypt fission and niche succession occur at a faster rate in adenoma tissue enabling accelerated clonal expansion. Methods: Fresh frozen human adenomas were obtained and clonal patches of adenomatous crypts identified by combining enzyme histochemistry and laser capture microdissection with the use of PCR sequencing to demonstrate shared clonal point mutations in mtDNA. Using DNA from the same crypt, methylation patterns at CpG islands of 3 non-expressed genes were determined using clonal bisulphite sequencing. Statistical analysis was then performed to compare crypt methylation patterns. Results: In contrast to normal human colon tissue, multiple, large clonal patches of crypts that shared common point mutations in their mtDNA were identified within human adenomas; these recently related adenomatous crypts had very similar, conserved methylation patterns compared to unrelated adenomatous crypts (p=8.08 x10-12). Conclusions: This study demonstrates that increased rates of niche succession, monoclonal conversion and crypt fission enable accelerated clonal expansion of mutated crypts in human adenomas, suggesting that these up-regulated processes allow rapid fixation and spread of successive oncogenic mutations during early tumorigenesis; further data collection will allow the time course of these events to be estimated. Combining the techniques used here with clonal oncogenic changes may reveal the selective growth advantage of specific mutations within adenomas. References: 1) Yatabe Y, Tavaré S, & Shibata D (2001). Investigating stem
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AGA Abstracts