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Insulin-Like Growth Factor-I Promotes Intestinal Stem Cell Expansion During Crypt Regeneration and Mucosal Healing Following Radiation
after irradiation and that IGF-I enhances these effects. Methods: Sox9-EGFP mice were given IGF-I (5μg/g body weight/day) or vehicle immediately after 14Gy abdominal irradiation and compared with non-irradiated controls. Histology and flow cytometry assessed Sox9-EGFP ISC between days 3 and 9 after irradiation. Expression of the type I IGF receptor (IGF-IR) was studied by immunohistochemistry and real-time qPCR on FACS-isolated cells. Results: Irradiation induced complete crypt loss by day 3. By day 4, there was massive expansion of Sox9-EGFP ISC, with a maximum 4.5 ±0.4 fold increase in ISC at day 5 and a return toward baseline by day 9. Co-localization of Sox9-EGFP and EdU, a proliferation marker, revealed that at day 5 after radiation, the proportion of proliferating Sox9-EGFP ISC was 2.8 ±0.2 fold higher than in non-irradiated controls. IGF-I treatment significantly increased Sox9-EGFP ISC by 1.7 ±0.2 fold in non-irradiated mice and further enhanced the maximum post-radiation induced increase in regenerating Sox9-EGFP ISC (+6.6 ±0.6 vs. non-irradiated controls). IGF-IR immunoreactivity was highly localized to crypt based columnar cells/ISC. IGF-IR mRNA expression was 9.4 ±1.0 fold higher in Sox9-EGFP-sorted ISC than in unsorted cells. Conclusions: We conclude that after radiation, Sox9-EGFP ISC expand and have direct roles in crypt regeneration. IGF-I potently and preferentially increases ISC numbers in normal or irradiated intestine, likely by signaling through IGF-IR whose expression is highly up-regulated in ISC. Ongoing microarray analyses are defining the molecular phenotype of regenerating or IGF-I stimulated ISC. IGF-I or hormones that induce IGF-I may promote ISC expansion in patients at risk of intestinal failure.
Lrig1 Marks a Distinct Population of Proliferative and Quiescent Intestinal Stem Cells and Acts as a Tumor Suppressor Anne Powell, Yina Li, Yang Wang, Jeff Franklin, Kevin Haigis, Robert J. Coffey Whether proliferative and quiescent intestinal stem cells coexist in normal and cancerous tissue is controversial. Due to heightened transcript expression in the intestinal epithelial stem cell compartment and detection in quiescent hair follicle stem cells, we hypothesized that Leucine-rich repeats and immunoglobulin-like domain protein 1 (Lrig1) would mark a novel population of intestinal stem cells and may be important for intestinal homeostasis and in cancer. To this end, we generated Lrig1-CreERT2/+; R26R-LacZ mice for Lrig1 intestinal epithelial cell lineage analysis. Examining adult mice at baseline and after irradiation damage, we show that Lrig1 labels both quiescent and proliferative stem cells and these cells are distinct from the well-defined Lgr5-expressing proliferative stem cell population. In addition, in a regenerative response to gut injury, these singly-labeled Lrig1-expressing cells proliferated and gave rise to clusters of labeled daughter cells, demonstrating the ability of these quiescent stem cells to become activated. Interestingly, loss of Lrig1 resulted in heightened expression of ErbB1-3 in the normal intestine, duodenal adenomas and carcinoma, supporting a role for Lrig1 in the maintenance of intestinal epithelial homeostasis and its ability to act as a tumor suppressor. To directly compare the role of the Lrig1- and Lgr5expressing cell populations in tumorigenesis, we used both Lrig1-CreERT2/+;Apcfl/fl and Lgr5-EGFP-IRES-CreERT2;Apcfl/fl mice to delete Apc. Five days after deletion, Lrig1CreERT2/+;Apcfl/fl mice have increased duodenal size and marked histological changes, which are absent in the Lgr5-EGFP-IRES-CreERT2;Apcfl/fl mice, further supporting a distinct and important role for the Lrig1 population in maintaining intestinal homeostasis. Finally, stochastic loss of Apc in Lrig1-expressing cells in Lrig1-CreERT2/+;Apcfl/+ mice results in multiple, large distal colonic tumors and less frequent, smaller intestinal tumors- common features of human familial polyposis- 4.5 months after Cre activation. In summary, we show that Lrig1 is a distinct marker of both proliferative and quiescent intestinal stem cells, is important for intestinal homeostasis and acts as a tumor suppressor.
162 Fusion of MSC With Gastric Epithelial Cells Increases Invasion and Metastasis of Gastric Cancer Hanchen Li, Calin Stoicov, Jian Hua Liu, Jean Marie Houghton Background The C57BL/6 mouse is a model of Hf induced gastric cancer. Infection causes gastritis, atrophy, metaplasia, dysplasia and gastric intraepithelial neoplasia (GIN). Prior studies from our group have shown that bone marrow (BM) derived mesenchymal stem cells (MSC) home into areas of inflammation, engraft into the mucosa and are the cell of origin of gastric cancer. In this study, we address what role fusion has in this process, and define the fusion partner within the gastric mucosa, and address the implication of fusion between epithelium and MSC carrying p53 mutations. Third, we determine if recruitment occurs at one point during infection or if it is an ongoing process. Methods P53MSC and wt MSC have been isolated and characterized by our laboratory. MSC populations were stably transfected with a modified ZEG construct. MSC ZEG are RFP/beta galactosidease positive in the absence of fusion, and after fusion with a cre-recombinase expressing cell, both RFP and GFP positive. Mice used were wt (no cre expression), K19-cre (expression of cre is limited to GI epithelial cells) and CMV-cre (all cell types express cre) transgenic mice. We infected mice with H.felis according to our standard protocol. At 9 months mice received a single iv injection of 1 x 106 MSC. 8 weeks later (11 months of infection) all mice were euthanized, stomachs removed, weighed, and processed for histology, immunofluorescence and immunohistochemisty. Full necropsy was performed. Results At 11 months of infection, wt, CMV-cre and K-19 cre mice +/- wt MSC had similar histological findings. Findings included chronic inflammation, mucus cell metaplasia and parietal cell loss. No mouse had dysplasia evident and there was no extra-gastric disease noted. Infected wt, CMV-cre and K19-Cre mice which had received p53 MSC containing the ZEG construct had widespread atrophy, dysplasia, invasion of glands into the submucosal area and apparent invasion of cells into the gastric vasculature. 50% of the mice (10/20) had multiple pulmonary nodules consistent with metastatic disease. The majority of dysplastic glands within the stomach were RFP+ thus defining them as originating from MSC cells. All of the RFP+ cells expressed GFP in both the K19 and CMV-cre model, (but not in the wt model) suggesting fusion between the MSC and the epithelial cell occurred. An epithelial phenotype was further confirmed by cytokeratin IHC. Lung nodules from K19 cre mice receiving p53MSC-ZEG were epithelial tumors with abundant stroma which stained positive for GFP, RFP and cytokeratin by specific IHC. Conclusions MSC are recruited to the Hf infected gastric mucosa on an on-going basis where they fuse with existing neoplastic and preneoplastic epithelial cells. Fusion between p53MSC and metaplastic epithelial cells increases the progression of local disease and lung metastasis.
160 CDC42 is Required for Intestinal Stem Cell Self-Renewal but Not Differentiation Lei Chen, Gene LeSage Adult intestinal stem cells (ISCs) possess long term self-renewal and multilineage differentiation properties to maintain intestinal epithelium. CDC42, a member of the Rho GTPase family, determines cell polarity by regulating actin, microtubule and protein distribution through Par6/aPKC signaling. Cdc42 is involved in the regulation of cell polarity, niche interaction, differentiation and proliferation of hematopoietic, neural and epidermis stem cells. Aims: To determine the effect of CDC42 loss of function on ISC self-renewal and differentiation and cell proliferation and migration along the crypt-villus axis. Methods: We employed an tamoxifen-inducible conditional gene targeting approach (Cdc42loxp/ loxp;CK19CreERT;Rosa-Stop-YFP mice) where loss of CDC42 and expression of YPF only occurs after the administration of tamoxifen and in CK19 expressing cells. For control studies, we used CK19CreERT;Rosa-stop-YFP mice. Cell polarity was determined by immunofluorescence microscopy for Zo-1, actin and beta-Catenin, proliferation by BrdU labeling and Ki76 staining, apoptosis by Caspase3 staining, and differentiation by FABP, MUC2, chromogranin and MMP7 staining. Mispositioning of cells was determined from EphrinB (villus) and EphB2 (crypt) distribution. Results: After the administration of tamoxifen, we demonstrated complete loss of CDC42 only in YFP+ cells. We found that Cdc42 deletion disrupted cell polarity and proliferation in the crypt (progenitor) cells but not villus (differentiated) cells without an increase in apoposis. Cell migration (determined by pulse BrdU labeling) and the expression of specific differentiation markers was the same in YPF positive and negative cells, indicating that CDC42 deficiency does not alter differentiation or migration. Administration of a single dose of tamoxifen to CK19CreERT;Rosa-stop-YFP mice resulted in long term retention (> 6 months) of YPF positive cells extending throughout the crypt-villus length, indicating permanent labeling of ISCs. Consistent with CDC42 being required for ISC self-renewal, we observed complete loss of YFP+, CDC42 deficient cells in Cdc42loxp/loxp;CK19CreERT;Rosa-Stop-YFP mice, first disappearing in the crypt by day 4 and then from the villus by day 10 following tamoxifen administration. Cdc42 deficient cells in the intervillus regions are EphrinB negative and EphB2 positive, which suggests CDC42 deficient cells mislocate to the intervillus region. There was a loss of the expression of Lrig1, a stem cell marker, and a decrease in pGSK3beta, pPKC, c-Myc and nuclear betaCatenin levels in the Cdc42 deficient crypt cells. Summary and conclusions: Our findings demonstrate an essential role of Cdc42 in self-renewal of intestinal stem/progenitor cells and retaining their localization in the crypts.
163 Deletion of Bak Leads to Expansion of the Stem Cell Niche in Murine Colonic Crypts Carrie A. Duckworth, Tanya Stezhka, David M. Pritchard Background: Intestinal epithelia are in a constant state of regeneration, sustained by stem cells which are thought to be located at or near the base of crypts. The intestinal stem cell niche is maintained by bidirectional signalling between epithelial stem cells and adjacent stromal pericryptal myofibroblasts. Bak is a pro-apoptotic bcl-2 family member that we have previously shown to be an important regulator of spontaneous and induced apoptosis, proliferation and differentiation in the colon. Reduced bak expression also increased susceptibility to colorectal carcinogenesis. We have currently investigated whether bak exerts these effects by altering the composition of the colonic stem cell niche. Methods: Tissue sections were prepared from formalin fixed and paraffin embedded or frozen samples of small intestine and colon from 10-12 week wild-type (C57BL/6) and bak-null mice. Haematoxylin and eosin staining was carried out to assess crypt morphology. Immunohistochemistry was performed for Ki67, DCAMKL-1 and CD24 (the latter two being potential stem cell markers in intestinal epithelia) and scored on a cell positional basis along the crypt axis. α-smooth muscle actin (α-SMA) immunohistochemistry was used to assess pericryptal myofibroblast numbers. Results: The colonic crypts of bak-null were significantly longer than those from C57BL/6 mice (bak-null, 33.5 ± 0.9; C57BL/6, 24.8 ± 0.3 cells per hemi-crypt; p<0.0001) and also showed a significant increase in Ki67 labelling index between cell positions 2-34. The percentage of Ki67 positive colonic epithelial cells was significantly increased in baknull mice compared with C57BL/6 mice, thus discounting the possibility that the observed differences were simply due to crypt hyperplasia (C57BL/6, 18.6 ± 2.3%; bak-null, 31.9 ± 1.9%, p<0.01). DCAMKL-1 and CD24 were expressed at the base and mid-crypt regions of colonic crypts in both bak-null and C57BL/6 mice, but bak-null mice showed significantly
161 Insulin-Like Growth Factor-I Promotes Intestinal Stem Cell Expansion During Crypt Regeneration and Mucosal Healing Following Radiation Laurianne Van Landeghem, Maria A. Santoro, Adrienne E. Krebs, Amanda T. Mah, Jeffrey J. Dehmer, Adam D. Gracz, Scott T. Magness, Pauline K. Lund High dose irradiation induces massive loss of intestinal epithelial crypts. After radiation, surviving intestinal stem cells (ISC) are thought to expand and regenerate the crypts. Insulinlike growth factor-I (IGF-I) has potent trophic effects on intestinal epithelium and mediates the actions of hormones used clinically to promote intestinal growth such as glucagon-like peptide-2 and growth hormone. Several studies indicate that IGF-I may exert selective or preferential anti-apoptotic effects on ISC. However, direct visualization of ISC regeneration after irradiation, or trophic effects of IGF-I on ISC, have not been demonstrated due to lack of ISC biomarkers. This study used a transgenic reporter mouse with EGFP expression driven by genomic regulatory sequences of the Sox9 gene which has been shown to mark multipotent ISC and permit their quantification or isolation (Gracz et al, Am J Physiol GI; 298(5):G590-600, 2010). We hypothesized that Sox9-EGFP ISC expand and regenerate crypts
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AGA Abstracts
AGA Abstracts
159
Lrig1 Marks a Distinct Population of Proliferative and Quiescent Intestinal Stem Cells and Acts as a Tumor Suppressor Anne Powell, Yina Li, Yang Wang, Jeff Franklin, Kevin Haigis, Robert J. Coffey Whether proliferative and quiescent intestinal stem cells coexist in normal and cancerous tissue is controversial. Due to heightened transcript expression in the intestinal epithelial stem cell compartment and detection in quiescent hair follicle stem cells, we hypothesized that Leucine-rich repeats and immunoglobulin-like domain protein 1 (Lrig1) would mark a novel population of intestinal stem cells and may be important for intestinal homeostasis and in cancer. To this end, we generated Lrig1-CreERT2/+; R26R-LacZ mice for Lrig1 intestinal epithelial cell lineage analysis. Examining adult mice at baseline and after irradiation damage, we show that Lrig1 labels both quiescent and proliferative stem cells and these cells are distinct from the well-defined Lgr5-expressing proliferative stem cell population. In addition, in a regenerative response to gut injury, these singly-labeled Lrig1-expressing cells proliferated and gave rise to clusters of labeled daughter cells, demonstrating the ability of these quiescent stem cells to become activated. Interestingly, loss of Lrig1 resulted in heightened expression of ErbB1-3 in the normal intestine, duodenal adenomas and carcinoma, supporting a role for Lrig1 in the maintenance of intestinal epithelial homeostasis and its ability to act as a tumor suppressor. To directly compare the role of the Lrig1- and Lgr5expressing cell populations in tumorigenesis, we used both Lrig1-CreERT2/+;Apcfl/fl and Lgr5-EGFP-IRES-CreERT2;Apcfl/fl mice to delete Apc. Five days after deletion, Lrig1CreERT2/+;Apcfl/fl mice have increased duodenal size and marked histological changes, which are absent in the Lgr5-EGFP-IRES-CreERT2;Apcfl/fl mice, further supporting a distinct and important role for the Lrig1 population in maintaining intestinal homeostasis. Finally, stochastic loss of Apc in Lrig1-expressing cells in Lrig1-CreERT2/+;Apcfl/+ mice results in multiple, large distal colonic tumors and less frequent, smaller intestinal tumors- common features of human familial polyposis- 4.5 months after Cre activation. In summary, we show that Lrig1 is a distinct marker of both proliferative and quiescent intestinal stem cells, is important for intestinal homeostasis and acts as a tumor suppressor.
162 Fusion of MSC With Gastric Epithelial Cells Increases Invasion and Metastasis of Gastric Cancer Hanchen Li, Calin Stoicov, Jian Hua Liu, Jean Marie Houghton Background The C57BL/6 mouse is a model of Hf induced gastric cancer. Infection causes gastritis, atrophy, metaplasia, dysplasia and gastric intraepithelial neoplasia (GIN). Prior studies from our group have shown that bone marrow (BM) derived mesenchymal stem cells (MSC) home into areas of inflammation, engraft into the mucosa and are the cell of origin of gastric cancer. In this study, we address what role fusion has in this process, and define the fusion partner within the gastric mucosa, and address the implication of fusion between epithelium and MSC carrying p53 mutations. Third, we determine if recruitment occurs at one point during infection or if it is an ongoing process. Methods P53MSC and wt MSC have been isolated and characterized by our laboratory. MSC populations were stably transfected with a modified ZEG construct. MSC ZEG are RFP/beta galactosidease positive in the absence of fusion, and after fusion with a cre-recombinase expressing cell, both RFP and GFP positive. Mice used were wt (no cre expression), K19-cre (expression of cre is limited to GI epithelial cells) and CMV-cre (all cell types express cre) transgenic mice. We infected mice with H.felis according to our standard protocol. At 9 months mice received a single iv injection of 1 x 106 MSC. 8 weeks later (11 months of infection) all mice were euthanized, stomachs removed, weighed, and processed for histology, immunofluorescence and immunohistochemisty. Full necropsy was performed. Results At 11 months of infection, wt, CMV-cre and K-19 cre mice +/- wt MSC had similar histological findings. Findings included chronic inflammation, mucus cell metaplasia and parietal cell loss. No mouse had dysplasia evident and there was no extra-gastric disease noted. Infected wt, CMV-cre and K19-Cre mice which had received p53 MSC containing the ZEG construct had widespread atrophy, dysplasia, invasion of glands into the submucosal area and apparent invasion of cells into the gastric vasculature. 50% of the mice (10/20) had multiple pulmonary nodules consistent with metastatic disease. The majority of dysplastic glands within the stomach were RFP+ thus defining them as originating from MSC cells. All of the RFP+ cells expressed GFP in both the K19 and CMV-cre model, (but not in the wt model) suggesting fusion between the MSC and the epithelial cell occurred. An epithelial phenotype was further confirmed by cytokeratin IHC. Lung nodules from K19 cre mice receiving p53MSC-ZEG were epithelial tumors with abundant stroma which stained positive for GFP, RFP and cytokeratin by specific IHC. Conclusions MSC are recruited to the Hf infected gastric mucosa on an on-going basis where they fuse with existing neoplastic and preneoplastic epithelial cells. Fusion between p53MSC and metaplastic epithelial cells increases the progression of local disease and lung metastasis.
160 CDC42 is Required for Intestinal Stem Cell Self-Renewal but Not Differentiation Lei Chen, Gene LeSage Adult intestinal stem cells (ISCs) possess long term self-renewal and multilineage differentiation properties to maintain intestinal epithelium. CDC42, a member of the Rho GTPase family, determines cell polarity by regulating actin, microtubule and protein distribution through Par6/aPKC signaling. Cdc42 is involved in the regulation of cell polarity, niche interaction, differentiation and proliferation of hematopoietic, neural and epidermis stem cells. Aims: To determine the effect of CDC42 loss of function on ISC self-renewal and differentiation and cell proliferation and migration along the crypt-villus axis. Methods: We employed an tamoxifen-inducible conditional gene targeting approach (Cdc42loxp/ loxp;CK19CreERT;Rosa-Stop-YFP mice) where loss of CDC42 and expression of YPF only occurs after the administration of tamoxifen and in CK19 expressing cells. For control studies, we used CK19CreERT;Rosa-stop-YFP mice. Cell polarity was determined by immunofluorescence microscopy for Zo-1, actin and beta-Catenin, proliferation by BrdU labeling and Ki76 staining, apoptosis by Caspase3 staining, and differentiation by FABP, MUC2, chromogranin and MMP7 staining. Mispositioning of cells was determined from EphrinB (villus) and EphB2 (crypt) distribution. Results: After the administration of tamoxifen, we demonstrated complete loss of CDC42 only in YFP+ cells. We found that Cdc42 deletion disrupted cell polarity and proliferation in the crypt (progenitor) cells but not villus (differentiated) cells without an increase in apoposis. Cell migration (determined by pulse BrdU labeling) and the expression of specific differentiation markers was the same in YPF positive and negative cells, indicating that CDC42 deficiency does not alter differentiation or migration. Administration of a single dose of tamoxifen to CK19CreERT;Rosa-stop-YFP mice resulted in long term retention (> 6 months) of YPF positive cells extending throughout the crypt-villus length, indicating permanent labeling of ISCs. Consistent with CDC42 being required for ISC self-renewal, we observed complete loss of YFP+, CDC42 deficient cells in Cdc42loxp/loxp;CK19CreERT;Rosa-Stop-YFP mice, first disappearing in the crypt by day 4 and then from the villus by day 10 following tamoxifen administration. Cdc42 deficient cells in the intervillus regions are EphrinB negative and EphB2 positive, which suggests CDC42 deficient cells mislocate to the intervillus region. There was a loss of the expression of Lrig1, a stem cell marker, and a decrease in pGSK3beta, pPKC, c-Myc and nuclear betaCatenin levels in the Cdc42 deficient crypt cells. Summary and conclusions: Our findings demonstrate an essential role of Cdc42 in self-renewal of intestinal stem/progenitor cells and retaining their localization in the crypts.
163 Deletion of Bak Leads to Expansion of the Stem Cell Niche in Murine Colonic Crypts Carrie A. Duckworth, Tanya Stezhka, David M. Pritchard Background: Intestinal epithelia are in a constant state of regeneration, sustained by stem cells which are thought to be located at or near the base of crypts. The intestinal stem cell niche is maintained by bidirectional signalling between epithelial stem cells and adjacent stromal pericryptal myofibroblasts. Bak is a pro-apoptotic bcl-2 family member that we have previously shown to be an important regulator of spontaneous and induced apoptosis, proliferation and differentiation in the colon. Reduced bak expression also increased susceptibility to colorectal carcinogenesis. We have currently investigated whether bak exerts these effects by altering the composition of the colonic stem cell niche. Methods: Tissue sections were prepared from formalin fixed and paraffin embedded or frozen samples of small intestine and colon from 10-12 week wild-type (C57BL/6) and bak-null mice. Haematoxylin and eosin staining was carried out to assess crypt morphology. Immunohistochemistry was performed for Ki67, DCAMKL-1 and CD24 (the latter two being potential stem cell markers in intestinal epithelia) and scored on a cell positional basis along the crypt axis. α-smooth muscle actin (α-SMA) immunohistochemistry was used to assess pericryptal myofibroblast numbers. Results: The colonic crypts of bak-null were significantly longer than those from C57BL/6 mice (bak-null, 33.5 ± 0.9; C57BL/6, 24.8 ± 0.3 cells per hemi-crypt; p<0.0001) and also showed a significant increase in Ki67 labelling index between cell positions 2-34. The percentage of Ki67 positive colonic epithelial cells was significantly increased in baknull mice compared with C57BL/6 mice, thus discounting the possibility that the observed differences were simply due to crypt hyperplasia (C57BL/6, 18.6 ± 2.3%; bak-null, 31.9 ± 1.9%, p<0.01). DCAMKL-1 and CD24 were expressed at the base and mid-crypt regions of colonic crypts in both bak-null and C57BL/6 mice, but bak-null mice showed significantly
161 Insulin-Like Growth Factor-I Promotes Intestinal Stem Cell Expansion During Crypt Regeneration and Mucosal Healing Following Radiation Laurianne Van Landeghem, Maria A. Santoro, Adrienne E. Krebs, Amanda T. Mah, Jeffrey J. Dehmer, Adam D. Gracz, Scott T. Magness, Pauline K. Lund High dose irradiation induces massive loss of intestinal epithelial crypts. After radiation, surviving intestinal stem cells (ISC) are thought to expand and regenerate the crypts. Insulinlike growth factor-I (IGF-I) has potent trophic effects on intestinal epithelium and mediates the actions of hormones used clinically to promote intestinal growth such as glucagon-like peptide-2 and growth hormone. Several studies indicate that IGF-I may exert selective or preferential anti-apoptotic effects on ISC. However, direct visualization of ISC regeneration after irradiation, or trophic effects of IGF-I on ISC, have not been demonstrated due to lack of ISC biomarkers. This study used a transgenic reporter mouse with EGFP expression driven by genomic regulatory sequences of the Sox9 gene which has been shown to mark multipotent ISC and permit their quantification or isolation (Gracz et al, Am J Physiol GI; 298(5):G590-600, 2010). We hypothesized that Sox9-EGFP ISC expand and regenerate crypts
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AGA Abstracts
AGA Abstracts
159