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925b Murine model for studying human intestine: Human Intestinal Organoids (HIOs) engrafted in vivo develop into mature epithelial and mesenchymal intestinal tissue
Methods: HIOs are generated in vitro from step-wise, directed differentiation of hPSCs. By modulating the BMP signaling pathway, the HIOs can also be patterned into a more proximal or distal intestinal phenotype. After 28 days, proximal and distal organoids contain epithelial and mesenchymal cell types that are highly similar to their in vivo equivalent segments of the gut. For engraftment in vivo, organoids cultured for 28–35 days are embedded into collagen and are then transplanted into the kidney capsule of immunocompromised mice. Six weeks following transplantation, the organoids are then harvested 2 hours following the administration of EdU and processed for histology and other analysis. Results: Successful engraftment occurred in a total of 19/35 (54%) proximal HIOs (Patterned with Noggin) and 24/35 (69%) distal HIOs (Patterned with EGF alone) revealing wellformed enteric-cystic structures which vary in size, but can be upwards of double the size of the mouse kidney. Histologically, human intestinal structures are maintained as demonstrated by CDX2 staining and human antinuclear antibodies. The mucosa is comprised of organized crypts and villi and contains all four intestinal epithelial lineages: enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. Positive EdU proliferative staining occurred at the base of crypts and within the mesenchyme. Immunohistochemistry confirmed both longitudinal and circular smooth muscle of human origin (using antibody to alphasmooth muscle actin and human anti-nuclear antibody). Capillary networks are also present and positive for human anti-nuclear antibody, however, most of the capillary networks are murine in origin. Lastly, immunohistochemistry for the enteric nervous system is currently in progress. Conclusions: This data demonstrates that HIOs from hPSCs can be efficiently transplanted in vivo and generate proliferating intestinal mucosa, mesenchyme, endothelium and muscle. This model provides a system to study human intestinal function, regionalization, adaptation, and congenital diseases in vivo. Methods to expand human intestine may also provide a potential source of bowel for tissue engineering and transplantation.
925a A Population of Lrig1–Expressing Basal Cells Act as Esophageal Stem Cells Gregory R. Vlacich, Robert J. Coffey Post-natal expansion and homeostasis of the stratified squamous epithelium in the mouse esophagus is maintained by a stem cell population thought to reside in the basal cell layer. Although various candidate stem cells markers have been proposed, none have yet been identified by long term lineage labeling. Lrig1, a negative regulator of the ErbB family of receptor tyrosine kinases, has recently been identified as a marker of largely quiescent, longlived intestinal and colonic stem cells (Cell 149:146–158, 2012). We now show that Lrig1 also marks a subset of basal cells in the adult and post-natal mouse esophagus that appear to function as stem cells. By lineage tracing in adult Lrig1CreERT2/+;ZSRosa26RLacZ/+ mice, Lrig1–expressing cells are present throughout the entire length of the esophagus. Two days after a single pulse of tamoxifen, labeled cells are initially scattered within the stratified squamous epithelium predominately as single or paired basal cells and constitute a small percentage of the proliferating basal cell population. With longer labeling up to 90 days, Lrig1+ cells give rise to suprabasal and superficial squamous cells, as well as additional basal cells, and this was confirmed with immunofluorescence co-localization studies. Notably, labeled cells were not observed in the mouse esophagus by short-term and long-term lineage tracing in Lgr5EGFP-Ires-CreERT2/+;ZSRosa26RLacZ/+ mice, suggesting this intestinal stem cell marker is not a marker in the esophagus. At post-natal day 1 (P1), Lrig1+ esophageal epithelial cells are also present and demonstrate robust expression within the basal layer as well as differentiation into the suprabasal layer in early post-natal development by lineage tracing. By early adulthood, the Lrig1–expressing population at P1 exhibits less suprabasal differentiation and by 180 days, this population of cells remains predominantly in contiguous basal cluster with little to no suprabasal staining. This is in stark contrast to the behavior of Lrig1–expressing cells in young adults in short- and long-term labeling, suggesting that these may be distinct populations potentially regulated by a distinct niche. These data are the first to demonstrate a squamous epithelial stem cell in the esophagus by lineage tracing and suggest that Lrig1 marks a subset of basal cells involved in esophageal growth and homeostasis. The divergent behavior of the P1 and adult Lrig1–expressing cells demonstrates a potentially novel dichotomy between stem-cell populations identified by a single marker and may help define the stem cell niche in the mouse esophagus. Additionally, injury and/ or genetic modification of this Lrig1+ basal cell population in adults may provide insight into a cell-of-origin for and critical genetic alterations in Barrett's esophagus and esophageal adenocarcinoma. (A) Cross-section of hPSC derived HIO at 6 weeks following transplantation- A single lumen is visible with intact villi (B) H&E staining of kidney with HIO transpant at 6 weeks posttransplantation (dotted line separates murine kidney above from engrafment below); Multiple areas are visible with epithelium surrounded by muscle layers and mesenchyme.
925b Murine model for studying human intestine: Human Intestinal Organoids (HIOs) engrafted in vivo develop into mature epithelial and mesenchymal intestinal tissue Carey L. Watson, Jonathan C. Howell, Jamie I. Schweitzer, Jorge Munera, Maxime M. Mahe, Nambirajan Sundaram, Jefferson Vallance, Noah F. Shroyer, James M. Wells, Michael A. Helmrath
925c A new Mendelian colorectal cancer predisposition syndrome caused by germline mutations in the proofreading domains of DNA polymerases epsilon and delta Ian Tomlinson
Background and Aims: Recent published work by Spence et al, Nature 2011, described in vitro methods for generating human intestinal organoids (HIOs) from both human embryonic stem cells and human induced pluripotent stem cells (collectively termed hPSCs), making the study of human intestinal tissue more feasible. In this study, we developed a murine model for in vivo study of human intestinal tissue via transplantation of HIOs. We hypothesized that the engrafted organoids will form all necessary layers of intestine and will proliferate to maintain the necessary cell types needed for functionality.
There exist patients with features of Mendelian colorectal cancer (CRC) syndromes - such as multiple tumours, early onset and family history of disease - but without identified mutations in the known CRC predisposition genes. We searched for new, high-penetrance predisposition genes in a set of cases and families with tens of colorectal adenomas and
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AGA Abstracts
AGA Abstracts
severity scores for nausea, early satiety, bloating, and upper abdominal pain; the minimum entry threshold was 2.5 (Day -14 to baseline [BL]). GSDD average change from baseline (CFB) through the end of treatment Weeks 11-12 was the primary endpoint and was compared between each TZP-102 dose vs. placebo using a rank ANCOVA test of superiority (1-sided α-0.025) with the Hochberg multiple comparison procedure. Additional outcomes included GCSI, PAGI-SYM, patient and physician Overall Treatment Evaluation (OTE), treatment-emergent AEs, and laboratory findings. Results: 201 patients (females 72%; Caucasians 87%; type 2 diabetes 61% with the following BL characteristics: on insulin 65%; age mean±SD 53±11.3 years; BMI 30.1±5.9; HbA1c 7.8±1.5%; GCSI 3.4±0.7; GSDD Composite 3.6±0.6; breath test [BT] half-emptying time 131±32 min) received study treatment (69, 66, and 66 patients received 10 mg, 20 mg TZP-102, and placebo, respectively). Treatment Results: No statistically significant improvements vs. placebo were observed in GSDD Composite score or GCSI for either TZP-102 dose (see table). In addition, the Week 12 mean patient OTE (3.9 [10 mg TZP-102]; 3.9 [20 mg TZP-102], and 3.3 placebo) and physician OTE scores (3.8, 3.7, 3.3, respectively) were not significantly different for TZP-102-treated patients vs. placebo. Both doses of TZP-102 were well tolerated; there were no study drugrelated serious AEs, no treatment-related pattern of Grade 3/4 laboratory abnormalities, and the most frequently observed AEs were in the gastrointestinal disorders system organ class as expected. Conclusions: Ghrelin agonist TZP-102 (orally QD) was well tolerated over 12 weeks. Symptoms of gastroparesis improved during treatment with 2 dose regimens, but there was no significant difference between either TZP-102 dose from placebo. TZP-102 Gastroparesis Symptom Measures (mean±SD)
925a A Population of Lrig1–Expressing Basal Cells Act as Esophageal Stem Cells Gregory R. Vlacich, Robert J. Coffey Post-natal expansion and homeostasis of the stratified squamous epithelium in the mouse esophagus is maintained by a stem cell population thought to reside in the basal cell layer. Although various candidate stem cells markers have been proposed, none have yet been identified by long term lineage labeling. Lrig1, a negative regulator of the ErbB family of receptor tyrosine kinases, has recently been identified as a marker of largely quiescent, longlived intestinal and colonic stem cells (Cell 149:146–158, 2012). We now show that Lrig1 also marks a subset of basal cells in the adult and post-natal mouse esophagus that appear to function as stem cells. By lineage tracing in adult Lrig1CreERT2/+;ZSRosa26RLacZ/+ mice, Lrig1–expressing cells are present throughout the entire length of the esophagus. Two days after a single pulse of tamoxifen, labeled cells are initially scattered within the stratified squamous epithelium predominately as single or paired basal cells and constitute a small percentage of the proliferating basal cell population. With longer labeling up to 90 days, Lrig1+ cells give rise to suprabasal and superficial squamous cells, as well as additional basal cells, and this was confirmed with immunofluorescence co-localization studies. Notably, labeled cells were not observed in the mouse esophagus by short-term and long-term lineage tracing in Lgr5EGFP-Ires-CreERT2/+;ZSRosa26RLacZ/+ mice, suggesting this intestinal stem cell marker is not a marker in the esophagus. At post-natal day 1 (P1), Lrig1+ esophageal epithelial cells are also present and demonstrate robust expression within the basal layer as well as differentiation into the suprabasal layer in early post-natal development by lineage tracing. By early adulthood, the Lrig1–expressing population at P1 exhibits less suprabasal differentiation and by 180 days, this population of cells remains predominantly in contiguous basal cluster with little to no suprabasal staining. This is in stark contrast to the behavior of Lrig1–expressing cells in young adults in short- and long-term labeling, suggesting that these may be distinct populations potentially regulated by a distinct niche. These data are the first to demonstrate a squamous epithelial stem cell in the esophagus by lineage tracing and suggest that Lrig1 marks a subset of basal cells involved in esophageal growth and homeostasis. The divergent behavior of the P1 and adult Lrig1–expressing cells demonstrates a potentially novel dichotomy between stem-cell populations identified by a single marker and may help define the stem cell niche in the mouse esophagus. Additionally, injury and/ or genetic modification of this Lrig1+ basal cell population in adults may provide insight into a cell-of-origin for and critical genetic alterations in Barrett's esophagus and esophageal adenocarcinoma. (A) Cross-section of hPSC derived HIO at 6 weeks following transplantation- A single lumen is visible with intact villi (B) H&E staining of kidney with HIO transpant at 6 weeks posttransplantation (dotted line separates murine kidney above from engrafment below); Multiple areas are visible with epithelium surrounded by muscle layers and mesenchyme.
925b Murine model for studying human intestine: Human Intestinal Organoids (HIOs) engrafted in vivo develop into mature epithelial and mesenchymal intestinal tissue Carey L. Watson, Jonathan C. Howell, Jamie I. Schweitzer, Jorge Munera, Maxime M. Mahe, Nambirajan Sundaram, Jefferson Vallance, Noah F. Shroyer, James M. Wells, Michael A. Helmrath
925c A new Mendelian colorectal cancer predisposition syndrome caused by germline mutations in the proofreading domains of DNA polymerases epsilon and delta Ian Tomlinson
Background and Aims: Recent published work by Spence et al, Nature 2011, described in vitro methods for generating human intestinal organoids (HIOs) from both human embryonic stem cells and human induced pluripotent stem cells (collectively termed hPSCs), making the study of human intestinal tissue more feasible. In this study, we developed a murine model for in vivo study of human intestinal tissue via transplantation of HIOs. We hypothesized that the engrafted organoids will form all necessary layers of intestine and will proliferate to maintain the necessary cell types needed for functionality.
There exist patients with features of Mendelian colorectal cancer (CRC) syndromes - such as multiple tumours, early onset and family history of disease - but without identified mutations in the known CRC predisposition genes. We searched for new, high-penetrance predisposition genes in a set of cases and families with tens of colorectal adenomas and
S-161
AGA Abstracts
AGA Abstracts
severity scores for nausea, early satiety, bloating, and upper abdominal pain; the minimum entry threshold was 2.5 (Day -14 to baseline [BL]). GSDD average change from baseline (CFB) through the end of treatment Weeks 11-12 was the primary endpoint and was compared between each TZP-102 dose vs. placebo using a rank ANCOVA test of superiority (1-sided α-0.025) with the Hochberg multiple comparison procedure. Additional outcomes included GCSI, PAGI-SYM, patient and physician Overall Treatment Evaluation (OTE), treatment-emergent AEs, and laboratory findings. Results: 201 patients (females 72%; Caucasians 87%; type 2 diabetes 61% with the following BL characteristics: on insulin 65%; age mean±SD 53±11.3 years; BMI 30.1±5.9; HbA1c 7.8±1.5%; GCSI 3.4±0.7; GSDD Composite 3.6±0.6; breath test [BT] half-emptying time 131±32 min) received study treatment (69, 66, and 66 patients received 10 mg, 20 mg TZP-102, and placebo, respectively). Treatment Results: No statistically significant improvements vs. placebo were observed in GSDD Composite score or GCSI for either TZP-102 dose (see table). In addition, the Week 12 mean patient OTE (3.9 [10 mg TZP-102]; 3.9 [20 mg TZP-102], and 3.3 placebo) and physician OTE scores (3.8, 3.7, 3.3, respectively) were not significantly different for TZP-102-treated patients vs. placebo. Both doses of TZP-102 were well tolerated; there were no study drugrelated serious AEs, no treatment-related pattern of Grade 3/4 laboratory abnormalities, and the most frequently observed AEs were in the gastrointestinal disorders system organ class as expected. Conclusions: Ghrelin agonist TZP-102 (orally QD) was well tolerated over 12 weeks. Symptoms of gastroparesis improved during treatment with 2 dose regimens, but there was no significant difference between either TZP-102 dose from placebo. TZP-102 Gastroparesis Symptom Measures (mean±SD)