- Email: [email protected]
199 Regulation of BMP-4 Expression by Inflammatory Cytokines
AGA Abstracts
the expression of its downstream target aftiphilin (AFPTH) (DDW2013: #934). Here we elucidated the mechanism by which miR-133α regulates NTS signaling in vitro, and studied the functional consequences of this response in colitis. Methods: NTS/miR-133α -regulated proinflammatory cytokine production in NCM460-NTSR1 cells and colon tissues was examined by qPCR and Bio-Plex Pro Human Cytokine Assay. Acute colonic inflammation was induced by intracolonic administration of TNBS (5 mg/kg, 48 h). Expression of miR-133α in the colon was inhibited by intracolonic administration of anti-sense (as)-miR-133α (2 doses, every two days) before TNBS treatment. The degree of inflammation was evaluated on distal colon segments stained with H&E. Results: MiR-133α overexpression in NCM460NTSR1 cells increased transcription of IL-8, IL-1β and TNF-α, while its knock-down attenuated NTS-induced IL-8 and IL-6 transcription (P<0.01). Bio-plex cytokine assays showed that NTS/miR-133α/AFTPH interactions are directly involved in the production of IL-1β (P<0.05). MiR-133α knock-down in mouse colon reduced colonic cxcl1, lcn2 and TNF-α expression (P<0.05) and neutrophil infiltration (P<0.05) in response to intracolonic TNBS administration. MiR-133α levels we increased (15-times, p<0.0001) during TNBS-induced colitis. MiR-133α knock-down in vivo also improved mucosal integrity (P<0.05) and overall histological score (P<0.05), supported by increased expression of the colonic epithelial cell marker, krt20 (P<0.01). Importantly, AFTPH transcription levels were significantly lower (P=0.0104) while miR-133α levels were up-regulated in colon tissues from patients with ulcerative colitis (n=22), compared to control tissues (n-21). Conclusions: miR-133α/AFTPH interactions regulate NTS-stimulated proinflammatory response in human colonic epithelial cells. Silencing of miR-133α in the colon reduces cytokine expression and histologic damage and inflammation in mouse colitis. These results suggest that miR-133α/AFTPH interactions promote NTS-induced proinflammatory responses in the colonic mucosa. Targeting of miR133α in the colon may represent a novel form for treatment in Inflammatory Bowel Disease. Supported by NIH grant DK60729 (CP), the Blinder Research Foundation for Crohn's Disease (IKML), and the Crohn's and Colitis Foundation of America (KB).
mice exhibited severe dysplasia of glands present in the ulcerated tissue. Interestingly, the gastric mucosa of aged mice transplanted with gastric organoids 30 days post-injury resembled that of young mice. 2) YFP+ organoids engraft within the injured site: Following transplantation, YFP+ organoid-derived cells engrafted in the injured gastric tissue. Organoid-derived cells engrafted within the ulcer margin. In particular, the glands formed by the ulcer associated cell lineage (UACL) were YFP+. Seven days post-injury, YFP+ organoid-derived engrafted cells also expressed CD44. 3) Engrafted organoid-derived cells maintain stem cell properties: CD44+ single cells isolated by FACS from the gastric tissue of aged mice 30 days posttransplantation, gave rise to organoids in culture. When placed under hypoxic conditions, gastric organoids generated from aged mice had decreased expression of growth factors associated with gastric repair such as VEGF and TGFα, compared to organoids generated from young mouse stomachs. CONCLUSION: Transplantation of gastric organoids containing stem cells promotes repair in the aged stomach in response to injury. CD44 may mark a population of stem cells within UACLs that regulate the repair process. 201 BVES Loss Is Protective in Radiation Enteritis and Influences Intestinal Stem Cell Programs Vishruth K. Reddy, Bobak Parang, Shenika Poindexter, Caitlyn W. Barrett, Wei Ning, Amber Bradley, Elizabeth Harris, Yash A. Choksi, Rishi D. Naik, Mukul K. Mittal, Kshipra Singh, Rupesh Chaturvedi, Thomas Brand, Kay Washington, David M. Bader, Keith T. Wilson, Christopher S. Williams Background: Blood Vessel Epicardial Substance (BVES) is a tight-junction associated protein that is expressed in the intestine and regulates epithelial-to-mesenchymal transition. We have previously demonstrated that BVES regulates colonic epithelial proliferation and differentiation, with BVES loss resulting in hyperactive WNT signaling, but its role in the small intestine (SI) remains unexplored. Methods: To determine the role of BVES in SI integrity we used radiation-induced injury modeling. Baseline differences between WT and Bves-/mice were assessed via IHC. To test morbidity after radiation, WT and Bves-/-mice (n=14) were irradiated with 15 Gray (Gy). Daily weight changes, stool scores, and food/water intake were monitored. Separately, another cohort of mice (n=17) was irradiated with 12 Gy. Mice were sacrificed 96 hours post-irradiation and SI assessed for crypt viability. To examine the role of BVES in intestinal crypt development and survival, we harvested enteroids from WT and Bves-/-mice, performed baseline phenotypic analysis, and isolated RNA to examine differentiation. Enteroids were then harvested from WT and Bves-/-mice irradiated with 12 Gy to examine enteroid viability post-irradiation. Results: Bves-/-mice had significantly greater proliferative (7.2 ± 0.4 vs. 6.2 ± 0.2, p<0.05) and goblet cells (19.7 ± 1.0 vs. 12.2 ± 0.3, p<0.001) per SI crypt/villus unit. Examination of H&E stained sections revealed Bves-/-mice exhibited significantly greater crypt viability after 12 Gy (64.4 ± 3.7% vs. 42.5 ± 7.8%, p<0.05). After 15 Gy, Bves-/-mice demonstrated significantly decreased weight loss when compared to WT (85.1 ± 8.1% vs. 78.1 ± 2.7%, p<0.05). No differences in stool scores or food/water intake were observed. Bves-/-enteroids demonstrated increased plating efficiency (48.3 ± 1.5% vs. 11.8 ± 2.5%, p<0.01), increased frequency of "stem spheroids," (15.7 ± 1.7% vs. 4.1 ± 1.0%, p<0.01) and accelerated progression to branching enteroids (21.7 ± 0.5% vs. 4.4 ± 2.6%, p<0.01), suggesting that BVES influences intestinal stem cell programs. RT-PCR analysis revealed baseline increases in MUC2 (4.4 ± 0.1 vs. 1.3 ± 0.1, p<0.01) and CGA (2.7 ± 0.2 vs. 1.3 ± 0.2, p<0.01) mRNA levels in Bves-/-enteroids compared to WT, suggesting greater populations of differentiated cells. Enteroids harvested from Bves-/-mice after 12 Gy irradiation also demonstrated increased plating efficiency when compared to WT (6.9 ± 0.2% vs. 3.2 ± 0.4%, p<0.01), suggesting greater viability after irradiation. Conclusion: BVES regulates SI proliferation and lineage allocation, modifies small intestinal crypt viability after radiation, and influences intestinal stem cell programs. These studies suggest that BVES loss promotes crypt viability and decreased morbidity after radiationinduced injury and may serve a protective role in radiation enteritis.
199 Regulation of BMP-4 Expression by Inflammatory Cytokines Tuo Ji, Hidehiko Takabayashi, Maria Y. Mao, Andrea Todisco BACKGROUND/AIMS: We reported that inhibition of Bone Morphogenetic Protein (BMP) signaling enhances Helicobacter-induced gastric inflammation suggesting that the BMPs exert anti-inflammatory actions in the stomach. In this study we tested the hypothesis that inflammatory stimuli could regulate BMP-4 expression. METHODS: 3 month-old C57BL/ 6-BMP-4-β-gal/+ mice were used to identify the localization of BMP-4 expressing cells in the gastric mucosa. Transgenic expression of IFN-γ in the oxyntic mucosa was achieved by means of the H+/K+-ATPase β-subunit gene promoter (H+/K+-IFN-γ TG-mice). Expression of BMP-4 was assessed by X-Gal staining, immunohistochemistry (IHC) with anti-β-gal antibodies (abs), and by QRT-PCR. Morphology of the gastric mucosa was examined in sections stained with H&E. Identification of fibroblasts was achieved by IHC with anti-α smooth muscle (α-SMA) and anti-vimentin abs. The effect of cytokines on BMP-4 expression was investigated by incubation of fibroblast cells (primary canine fibroblasts, NL-685 human fibroblasts and CCD-18Co human myofibroblasts) with IFN-γ, TNF-α, IL-1β, and IL-6. Western blots were used to assess the phosphorylation and activation of STAT-1. Fibroblast proliferation was assessed by BrdU incorporation. RESULTS: Analysis of mucosal sections of C57BL/6-BMP-4-β-gal/+ mice stained with X-gal, demonstrated that BMP-4 is expressed in the mesenchymal layers of the gastric mucosa. BMP-4 staining was seen in α-SMA- and vimentin-positive cells. IFN-γ induced BMP-4 gene expression in canine fibroblasts, NL685 cells and in CCD-18Co myofibroblasts. Both TNF-α and IL-1β inhibited the expression of BMP-4 in CCD-18Co cells, while no significant effect was seen in the presence of IL-6. Incubation of CCD-18Co cells with IFN-γ in combination with TNF-α led to abrogation of the stimulatory effect of IFN-γ on BMP-4 gene expression. IFN-γ induced STAT-1 phosphorylation with a maximal effect seen between 5 and 60 min of incubation. Inhibition of the upstream STAT-1 activating kinase JAK1, blocked IFN-γ stimulation of BMP-4 gene expression. Overexpression of IFN-γ in the gastric mucosa of 8 week-old H+/K+-IFN-γ TG-mice, led to gastric inflammation and to a significant increase in BMP-4 gene expression. IFN-γ also induced the proliferation of canine gastric fibroblasts. CONCLUSIONS: BMP-4 is expressed in gastric fibroblasts. Inflammatory stimuli modulate the expression of BMP-4 in fibroblast cells. IFN-γ appears to stimulate BMP-4 expression both in vitro and in vivo. The actions of IFN-γ involve the activation of JAK/STAT-1-dependent pathways. These effects might represent a self-safe mechanism that tampers the intensity of the inflammatory response.
202 Intestinal Region-Specific Pattern Is Borne and Maintained Within the Intestinal Stem Cells Maxime M. Mahe, Yuan-Hung Lo, Carey L. Watson, Nambirajan Sundaram, Fengchao Wang, Rebekah Karns, Joo-Seop Park, Linheng Li, Noah F. Shroyer, Michael A. Helmrath Regional differences in small intestinal physiology exist along the rostro-caudal axis as demonstrated by distinct morphological and digestive differences. The proximal small bowel expresses region-specific digestive enzymes and is the primary site for water-soluble vitamins and iron absorption. The distal small bowel is the site for the absorption of bile acids and vitamin B12. Clinically, these differences are evident in common diseases that occur more frequently in the distal small bowel such as Crohn's disease, and enhanced ileum adaptation seen following surgical resection. These observations strongly suggest that a specific regional intestinal epithelial patterning exists. Unravelling the cellular and molecular basis for intestinal regional identity (rID), this study addresses the hypothesis that murine and human intestinal stem cells (ISCs) support a region-specific pattern. We screened regional gene expression from murine crypts, ISCs and cultured enteroids. RNA sequencing was performed on isolated murine crypts, FACS-sorted Lgr5-GFPHi and CD44+/CD24lo/CD166+ ISCs. In addition, histone ChIP-sequencing (H3K27Ac and H3K27me3) were performed on jejunal and ileal crypts. Both Lgr5-GFPHi and CD44+/CD24lo/CD166+ single cells derived proximal and distal enteroids were generated and screened against regional genes. Finally, human isolated crypts and cultured enteroids were screened against the same gene expression panel. Each experiment was performed with n=4-6 per group. Proximal intestinal markers, i.e. Gata4, Onecut2 and Pdx1, and distal markers,Adcy8, Fabp6 and Prss23 remained enriched for their respective region in enteroids following multiple passages. RNA sequencing showed that regional ISCs populations were differentially enriched for proximal and distal genes. We found open chromatin marks (H3K27Ac) encompassing genes enriched in the jejunum including Pdx1, Gata4, and Onecut2, whereas closed chromatin marks (H3K27me3) were associated with distal genes including Adcy8 and Prss23, The opposite pattern was observed for the ileum. We demonstrate that isolated Lgr5-GFPHi and CD44+/CD24lo/CD166+ single cells form proximal jejunal and distal ileal enteroids and maintained appropriate expression
200 Engraftment of Stem Cells Derived From Gastric Organoids Promotes Ulcer Repair in the Aged Stomach Amy C. Engevik, Rui Feng, Eitaro Aihara, Marshall H. Montrose, Yana Zavros BACKGROUND: Advancing age contributes to an increased susceptibility to gastric injury and decreased capacity to repair damaged gastric tissue. This results in an increased incidence of peptic ulcers in the elderly. During gastric ulcer repair epithelial stem cells from the base of the ulcer margin invade granulation tissue, proliferate and differentiate into gastric glands within the injured site. The hyaluronan receptor CD44 has been identified as a potential stem cell marker of the gastric epithelium. The expression and role of CD44 during gastric repair is unknown. HYPOTHESIS: Engraftment of stem cells, through the transplantation of gastric organoids in aged mice promotes normal ulcer repair. METHODS: Ulcers were induced in C57BL/6 mice by applying acetic acid on the serosa of the stomach. Mice were grouped according to age: young 2-3 months, middle aged 10-14 months and aged 18-24 months old. Gastric organoids were generated from yellow-emitting fluorescent protein YFP transgenic mouse stomachs (YFP+ organoids). YFP+ gastric organoids cultured in vitro were transplanted after ulcer induction into the lumen and the submucosa of the stomach. Gastric tissue was collected and analyzed for repair and engraftment of YFP+ cells. YFP+ cells were isolated from transplanted stomachs by FACS and grown in culture. RESULTS: 1) Transplantation of gastric organoids promotes gastric repair in the aged stomach: In mice aged 2-3 months ulcers healed within 30 days post-injury. H&E staining showed re-epithelialization of damaged gastric tissue with normal gastric gland architecture. In contrast, aged
AGA Abstracts
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the expression of its downstream target aftiphilin (AFPTH) (DDW2013: #934). Here we elucidated the mechanism by which miR-133α regulates NTS signaling in vitro, and studied the functional consequences of this response in colitis. Methods: NTS/miR-133α -regulated proinflammatory cytokine production in NCM460-NTSR1 cells and colon tissues was examined by qPCR and Bio-Plex Pro Human Cytokine Assay. Acute colonic inflammation was induced by intracolonic administration of TNBS (5 mg/kg, 48 h). Expression of miR-133α in the colon was inhibited by intracolonic administration of anti-sense (as)-miR-133α (2 doses, every two days) before TNBS treatment. The degree of inflammation was evaluated on distal colon segments stained with H&E. Results: MiR-133α overexpression in NCM460NTSR1 cells increased transcription of IL-8, IL-1β and TNF-α, while its knock-down attenuated NTS-induced IL-8 and IL-6 transcription (P<0.01). Bio-plex cytokine assays showed that NTS/miR-133α/AFTPH interactions are directly involved in the production of IL-1β (P<0.05). MiR-133α knock-down in mouse colon reduced colonic cxcl1, lcn2 and TNF-α expression (P<0.05) and neutrophil infiltration (P<0.05) in response to intracolonic TNBS administration. MiR-133α levels we increased (15-times, p<0.0001) during TNBS-induced colitis. MiR-133α knock-down in vivo also improved mucosal integrity (P<0.05) and overall histological score (P<0.05), supported by increased expression of the colonic epithelial cell marker, krt20 (P<0.01). Importantly, AFTPH transcription levels were significantly lower (P=0.0104) while miR-133α levels were up-regulated in colon tissues from patients with ulcerative colitis (n=22), compared to control tissues (n-21). Conclusions: miR-133α/AFTPH interactions regulate NTS-stimulated proinflammatory response in human colonic epithelial cells. Silencing of miR-133α in the colon reduces cytokine expression and histologic damage and inflammation in mouse colitis. These results suggest that miR-133α/AFTPH interactions promote NTS-induced proinflammatory responses in the colonic mucosa. Targeting of miR133α in the colon may represent a novel form for treatment in Inflammatory Bowel Disease. Supported by NIH grant DK60729 (CP), the Blinder Research Foundation for Crohn's Disease (IKML), and the Crohn's and Colitis Foundation of America (KB).
mice exhibited severe dysplasia of glands present in the ulcerated tissue. Interestingly, the gastric mucosa of aged mice transplanted with gastric organoids 30 days post-injury resembled that of young mice. 2) YFP+ organoids engraft within the injured site: Following transplantation, YFP+ organoid-derived cells engrafted in the injured gastric tissue. Organoid-derived cells engrafted within the ulcer margin. In particular, the glands formed by the ulcer associated cell lineage (UACL) were YFP+. Seven days post-injury, YFP+ organoid-derived engrafted cells also expressed CD44. 3) Engrafted organoid-derived cells maintain stem cell properties: CD44+ single cells isolated by FACS from the gastric tissue of aged mice 30 days posttransplantation, gave rise to organoids in culture. When placed under hypoxic conditions, gastric organoids generated from aged mice had decreased expression of growth factors associated with gastric repair such as VEGF and TGFα, compared to organoids generated from young mouse stomachs. CONCLUSION: Transplantation of gastric organoids containing stem cells promotes repair in the aged stomach in response to injury. CD44 may mark a population of stem cells within UACLs that regulate the repair process. 201 BVES Loss Is Protective in Radiation Enteritis and Influences Intestinal Stem Cell Programs Vishruth K. Reddy, Bobak Parang, Shenika Poindexter, Caitlyn W. Barrett, Wei Ning, Amber Bradley, Elizabeth Harris, Yash A. Choksi, Rishi D. Naik, Mukul K. Mittal, Kshipra Singh, Rupesh Chaturvedi, Thomas Brand, Kay Washington, David M. Bader, Keith T. Wilson, Christopher S. Williams Background: Blood Vessel Epicardial Substance (BVES) is a tight-junction associated protein that is expressed in the intestine and regulates epithelial-to-mesenchymal transition. We have previously demonstrated that BVES regulates colonic epithelial proliferation and differentiation, with BVES loss resulting in hyperactive WNT signaling, but its role in the small intestine (SI) remains unexplored. Methods: To determine the role of BVES in SI integrity we used radiation-induced injury modeling. Baseline differences between WT and Bves-/mice were assessed via IHC. To test morbidity after radiation, WT and Bves-/-mice (n=14) were irradiated with 15 Gray (Gy). Daily weight changes, stool scores, and food/water intake were monitored. Separately, another cohort of mice (n=17) was irradiated with 12 Gy. Mice were sacrificed 96 hours post-irradiation and SI assessed for crypt viability. To examine the role of BVES in intestinal crypt development and survival, we harvested enteroids from WT and Bves-/-mice, performed baseline phenotypic analysis, and isolated RNA to examine differentiation. Enteroids were then harvested from WT and Bves-/-mice irradiated with 12 Gy to examine enteroid viability post-irradiation. Results: Bves-/-mice had significantly greater proliferative (7.2 ± 0.4 vs. 6.2 ± 0.2, p<0.05) and goblet cells (19.7 ± 1.0 vs. 12.2 ± 0.3, p<0.001) per SI crypt/villus unit. Examination of H&E stained sections revealed Bves-/-mice exhibited significantly greater crypt viability after 12 Gy (64.4 ± 3.7% vs. 42.5 ± 7.8%, p<0.05). After 15 Gy, Bves-/-mice demonstrated significantly decreased weight loss when compared to WT (85.1 ± 8.1% vs. 78.1 ± 2.7%, p<0.05). No differences in stool scores or food/water intake were observed. Bves-/-enteroids demonstrated increased plating efficiency (48.3 ± 1.5% vs. 11.8 ± 2.5%, p<0.01), increased frequency of "stem spheroids," (15.7 ± 1.7% vs. 4.1 ± 1.0%, p<0.01) and accelerated progression to branching enteroids (21.7 ± 0.5% vs. 4.4 ± 2.6%, p<0.01), suggesting that BVES influences intestinal stem cell programs. RT-PCR analysis revealed baseline increases in MUC2 (4.4 ± 0.1 vs. 1.3 ± 0.1, p<0.01) and CGA (2.7 ± 0.2 vs. 1.3 ± 0.2, p<0.01) mRNA levels in Bves-/-enteroids compared to WT, suggesting greater populations of differentiated cells. Enteroids harvested from Bves-/-mice after 12 Gy irradiation also demonstrated increased plating efficiency when compared to WT (6.9 ± 0.2% vs. 3.2 ± 0.4%, p<0.01), suggesting greater viability after irradiation. Conclusion: BVES regulates SI proliferation and lineage allocation, modifies small intestinal crypt viability after radiation, and influences intestinal stem cell programs. These studies suggest that BVES loss promotes crypt viability and decreased morbidity after radiationinduced injury and may serve a protective role in radiation enteritis.
199 Regulation of BMP-4 Expression by Inflammatory Cytokines Tuo Ji, Hidehiko Takabayashi, Maria Y. Mao, Andrea Todisco BACKGROUND/AIMS: We reported that inhibition of Bone Morphogenetic Protein (BMP) signaling enhances Helicobacter-induced gastric inflammation suggesting that the BMPs exert anti-inflammatory actions in the stomach. In this study we tested the hypothesis that inflammatory stimuli could regulate BMP-4 expression. METHODS: 3 month-old C57BL/ 6-BMP-4-β-gal/+ mice were used to identify the localization of BMP-4 expressing cells in the gastric mucosa. Transgenic expression of IFN-γ in the oxyntic mucosa was achieved by means of the H+/K+-ATPase β-subunit gene promoter (H+/K+-IFN-γ TG-mice). Expression of BMP-4 was assessed by X-Gal staining, immunohistochemistry (IHC) with anti-β-gal antibodies (abs), and by QRT-PCR. Morphology of the gastric mucosa was examined in sections stained with H&E. Identification of fibroblasts was achieved by IHC with anti-α smooth muscle (α-SMA) and anti-vimentin abs. The effect of cytokines on BMP-4 expression was investigated by incubation of fibroblast cells (primary canine fibroblasts, NL-685 human fibroblasts and CCD-18Co human myofibroblasts) with IFN-γ, TNF-α, IL-1β, and IL-6. Western blots were used to assess the phosphorylation and activation of STAT-1. Fibroblast proliferation was assessed by BrdU incorporation. RESULTS: Analysis of mucosal sections of C57BL/6-BMP-4-β-gal/+ mice stained with X-gal, demonstrated that BMP-4 is expressed in the mesenchymal layers of the gastric mucosa. BMP-4 staining was seen in α-SMA- and vimentin-positive cells. IFN-γ induced BMP-4 gene expression in canine fibroblasts, NL685 cells and in CCD-18Co myofibroblasts. Both TNF-α and IL-1β inhibited the expression of BMP-4 in CCD-18Co cells, while no significant effect was seen in the presence of IL-6. Incubation of CCD-18Co cells with IFN-γ in combination with TNF-α led to abrogation of the stimulatory effect of IFN-γ on BMP-4 gene expression. IFN-γ induced STAT-1 phosphorylation with a maximal effect seen between 5 and 60 min of incubation. Inhibition of the upstream STAT-1 activating kinase JAK1, blocked IFN-γ stimulation of BMP-4 gene expression. Overexpression of IFN-γ in the gastric mucosa of 8 week-old H+/K+-IFN-γ TG-mice, led to gastric inflammation and to a significant increase in BMP-4 gene expression. IFN-γ also induced the proliferation of canine gastric fibroblasts. CONCLUSIONS: BMP-4 is expressed in gastric fibroblasts. Inflammatory stimuli modulate the expression of BMP-4 in fibroblast cells. IFN-γ appears to stimulate BMP-4 expression both in vitro and in vivo. The actions of IFN-γ involve the activation of JAK/STAT-1-dependent pathways. These effects might represent a self-safe mechanism that tampers the intensity of the inflammatory response.
202 Intestinal Region-Specific Pattern Is Borne and Maintained Within the Intestinal Stem Cells Maxime M. Mahe, Yuan-Hung Lo, Carey L. Watson, Nambirajan Sundaram, Fengchao Wang, Rebekah Karns, Joo-Seop Park, Linheng Li, Noah F. Shroyer, Michael A. Helmrath Regional differences in small intestinal physiology exist along the rostro-caudal axis as demonstrated by distinct morphological and digestive differences. The proximal small bowel expresses region-specific digestive enzymes and is the primary site for water-soluble vitamins and iron absorption. The distal small bowel is the site for the absorption of bile acids and vitamin B12. Clinically, these differences are evident in common diseases that occur more frequently in the distal small bowel such as Crohn's disease, and enhanced ileum adaptation seen following surgical resection. These observations strongly suggest that a specific regional intestinal epithelial patterning exists. Unravelling the cellular and molecular basis for intestinal regional identity (rID), this study addresses the hypothesis that murine and human intestinal stem cells (ISCs) support a region-specific pattern. We screened regional gene expression from murine crypts, ISCs and cultured enteroids. RNA sequencing was performed on isolated murine crypts, FACS-sorted Lgr5-GFPHi and CD44+/CD24lo/CD166+ ISCs. In addition, histone ChIP-sequencing (H3K27Ac and H3K27me3) were performed on jejunal and ileal crypts. Both Lgr5-GFPHi and CD44+/CD24lo/CD166+ single cells derived proximal and distal enteroids were generated and screened against regional genes. Finally, human isolated crypts and cultured enteroids were screened against the same gene expression panel. Each experiment was performed with n=4-6 per group. Proximal intestinal markers, i.e. Gata4, Onecut2 and Pdx1, and distal markers,Adcy8, Fabp6 and Prss23 remained enriched for their respective region in enteroids following multiple passages. RNA sequencing showed that regional ISCs populations were differentially enriched for proximal and distal genes. We found open chromatin marks (H3K27Ac) encompassing genes enriched in the jejunum including Pdx1, Gata4, and Onecut2, whereas closed chromatin marks (H3K27me3) were associated with distal genes including Adcy8 and Prss23, The opposite pattern was observed for the ileum. We demonstrate that isolated Lgr5-GFPHi and CD44+/CD24lo/CD166+ single cells form proximal jejunal and distal ileal enteroids and maintained appropriate expression
200 Engraftment of Stem Cells Derived From Gastric Organoids Promotes Ulcer Repair in the Aged Stomach Amy C. Engevik, Rui Feng, Eitaro Aihara, Marshall H. Montrose, Yana Zavros BACKGROUND: Advancing age contributes to an increased susceptibility to gastric injury and decreased capacity to repair damaged gastric tissue. This results in an increased incidence of peptic ulcers in the elderly. During gastric ulcer repair epithelial stem cells from the base of the ulcer margin invade granulation tissue, proliferate and differentiate into gastric glands within the injured site. The hyaluronan receptor CD44 has been identified as a potential stem cell marker of the gastric epithelium. The expression and role of CD44 during gastric repair is unknown. HYPOTHESIS: Engraftment of stem cells, through the transplantation of gastric organoids in aged mice promotes normal ulcer repair. METHODS: Ulcers were induced in C57BL/6 mice by applying acetic acid on the serosa of the stomach. Mice were grouped according to age: young 2-3 months, middle aged 10-14 months and aged 18-24 months old. Gastric organoids were generated from yellow-emitting fluorescent protein YFP transgenic mouse stomachs (YFP+ organoids). YFP+ gastric organoids cultured in vitro were transplanted after ulcer induction into the lumen and the submucosa of the stomach. Gastric tissue was collected and analyzed for repair and engraftment of YFP+ cells. YFP+ cells were isolated from transplanted stomachs by FACS and grown in culture. RESULTS: 1) Transplantation of gastric organoids promotes gastric repair in the aged stomach: In mice aged 2-3 months ulcers healed within 30 days post-injury. H&E staining showed re-epithelialization of damaged gastric tissue with normal gastric gland architecture. In contrast, aged
AGA Abstracts
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