$951

Fibroblast Growth Factors Promote Hepatocytc Stem Cell Renewal in Embryonic Liver Cultures Sandeep Sekhon, Xinping Tan, William Bowen, George Michalopoulos, Satdarshan Pal S. Monga

Fibroblast growth factors (FGFs) have been shown to play a definite role in hepatic induction. Stage and tissue specific expression of FGF 1,4 and 8 are necessary for initiation of hepatic bud from the foregut endoderm FGF is also routinely used to maintain and propagate embryonic stem ceils in culture. The aim of this study was to investigate the effect of FGF 1, 4 and 8 on liver development utilizing an in vitro embryonic liver culture system and recotnbinant FGF proteins. Livers were harvested from day 10 embryos (El0) and cultured as an organ in DMEM supplemented with 10% FCS (control) with or without additional FGF 1 or FGF 4 or FGF 8. After 72 hours, the cultured livers were fixed and processed and 4/zm sections were utilized for histologic and immunohistochemical evaluation. Proliferation (PCNA and Ki-67) and apoptosis (TUNEL) assay was also performed. The major difference in the histology of the FGF 1/4/8 treated cultures was in the arrangement of cells in sheet like architecture only as compared to the controls that displayed cells in both ductular and sheet like arrangements. There was no significant change in number of apoptotic cells in presence of FGF 1/4 but there was a decrease in number of apoptotic nuclei in FGF8 treated cultures. Almost all cells in all FGF cultures exhibited PCNA positivity and a sigmficant number of cells were Ki-67 (S-phase) positive. There was a striking difference in the number of c-kn positive stem cells in the presence of any of the three FGFs used as compared to the controls. These stem cells were also positive for a-fetoprotein and albumin and very few were positive for CK-19. There was an overall decrease in number of CK-19 positive cells in presence of the FGFs. To conclude, FGF 1, 4 and 8 are excellent growth factors to maintain and propagate liver stem cells in development. These factors support prohferation and affect apoptosis and thus promote liver stem cell renewal. Also, lineage analysis shows a preponderance of unipotential stem cells (more hepatocytic than biliary) in the FGF treated cultures that suggest existence of selective hepatocyte stem ceils, h appears that FGFs promotes self-renewal of such a unipotential stem cell population in the liver.

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Potential of Bone Marrow Cells for Epithelial Replacement Therapy in the Murine Gut Satish K. Singh, Selvi Kr/shnau, Daisuke Kohayashi, Hiroshi Mashimo

BACKGROUND/AIMS: There is evidence that hematogenously injected bone marrow stem cells may sporadically differentiate into gut epithelial cells. We hypothesized that a large number of bone marrow cells could differentiate into epithelial cells when injected locally in the adult murine gut. METHODS: Bone marrow ceils were harvested from femurs and tibias of transgenic ROSA26 mice (Jackson Labs). These ceils constitutively express beta- gaIactosidase which serves as a discernible marker after injection into host (C57BL/6Jx129SV/ J)F1 mice (Jackson Labs). The derived ROSA26 bone marrow cells were sypgeneic with our host mice, negating the need for immunosuppression. After panning on plastic petridish, non-adherent bone marrow cells (5 x 104 ceils) were microinjected into the gastric and proximal duodenal wall of 6-8 week old adult mice. These tissues were harvested at 1 and 4 weeks post-injection for immunohistochemical staining. RESULTS: Derived ROSA26 bone marrow cells stained strongly blue for beta-galactnsidase. These cells persisted in vivo at the injection site when assessed at 1 and 4 weeks. There was no associated inflammation or tumors. Moreover, injected ceils preferentially engrafted into the deeper regions of the gastric and duodenal mucosa. These cells formed some gland-like structures, and respected local tissue architecture. Beta-galactosidase positive cells within the mucosa expressed markers of epithelial differentiation including cytokeratin and e-cadherin. In addition, certain beta- galactosidase positive gastric epithelial cells stained positive for H + -K § -ATPase and the gastrin receptor. CONCLUSIONS: Bone marrow cells implanted into the stomach and duodenal wall engraft, and are capable of differeutiating into epithelial cells. This approach holds promise for long-term implantation and epithelial replacement, and may lead to treatments for a wide range of ulcerative, degenerative, and genetic diseases of the gut.

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Primary Cultures of Fully Differentiated Mouse Intestinal Mucosa Akifumi Ootani, Kazuma Fujimoto, Shuji Toda, Hajime Sugihara

The factors controlling the proliferation and differentiation of the intestinal mucosa are unknown and have proved difficult to identify because of a lack of in vitro models for studying the proliferating cells of the mucosa. The aim of this study was to develop a novel method for preparing a viable fully differentiated normal enterocytes. METHODS: Minced intestines of newborn mouse were cultured in three-dimensional collagen gel system at an air-liquid interface environment for 7 days. RESULTS: The polarized epithelial cells organized the tubule structure with outer n'tyofibroblast lining. The epithelial ceils consisted of absorp- tive enterocytes and goblet cells. The former showed a tall columnar cell shape with blush border and immunoreactivity for absorptive cell marker, CD10. The latter showed mucous containing cytoplasm with basal nuclei and positive staining for goblet cell marker, MUC2. 13romodeoxyuridine uptake study showed that active growth of these cells. Interestingly, the viable epithelial cells were observed invariably with underlying myofibroblasts. Tenascin- C, fibronectin-derived anti-adhesive peptide, was expressed in apical and basolateral side of the fully differentiated epithelial cells. When the tissue fragments were cultured under immersed conditions, none of the viable ceils were observed. CONCLUSIONS: This investiga- tion establishes for the first time that mouse intestinal mucosa can be maintained with fully differentiation in vitro. Our results suggest that intestinal myofibroblasts play a crucial role in the survival, proliferation, and differentiation of intestinal epithelial cells, and that the expression of tenascin-C may be involved in the regeneration and maintenance of the intestinal tissue architecture under epithehal-mesenchymal interaction.

$954

Paneth Ceils Secrete A Soluble Isoform Of Maltase-Glucoamylase (MGAs) With Full Starch Digesting Capacity In Membrane MGA (MGAm) Null Mice Buford L. Nichols Jr., Stephen E. Avery, Nancy F. Butte, Ursula Luginbuehl, Erwin E. Sterchi, Milton Finegold

Background: Although starch is the major energy substrate in the diet, nothing is known about the phenotype of adult MGA deficiency. MGA is the enzyme that converts the dominant 1-4 linkages of solubilized starch to glucose. We generated a knockout of MGAm to study MGA deficiency phenotype. Methods: A selection cassette containing Lac-Z was inserted in place of exon 2 of MGAm KO was confirmed by Q-PCR. X-gal was used to stain tissue Lac-Z expression. Glucoamylase activity (GA) was measured by Dahlqvist assays with Polycose substrate. Soluble forms were isolated by 100,000 G centrifugation. In vivo starch utilization was measured as prandial kCal/g/hr in calorimeters on amylopectin and amylose diets. lmmunohistology (IH) used affinity purified polyclonal antibodies against C-terminal. Maize fragments were identified by polarized light microscopy. KO null and Wildtype (WT)5' cDNA were sequenced. Results: Q-PCR and X-gal staining confirmed the MGAm KO. Jejunal GA was decreased by 40% but ileal GA was intact in KO nulls. 28% of ileal GA was in KO and WT homogenate supematants. Amylopectin and amylose utilizations were identical in KO null and WT by calorimetry. IH showed absence of enterocyte staining in KO nulls but intense staining of Paneth cell granules in KO and WT. Staining was faintly present in some goblets. No staining was detected in colonic mucosa. IH revealed abundant MGAs staining in ileal and colonic lumens. Washings of small intestine revealed GA in nulls and WT. Colonic null and WT homogenates had GA. 66% of small intestine luminal and colonic homogenate GA was in supernatants. By IH and polarized light, infranatant ileal lumen MGAs was bound to starch granules and maize cell fragments. The KO null cDNA sequence revealed excisional splicing of MGAm exon 2, which codes the binding domain. Conclusion: KO of MGAm revealed the presence of MGKs, a secreted isoform, produced by Paneth cells. The MGAs activity was sufficient to maintain normal starch utilization in KO nulls. MGAs was present by IH in ileal and colonic lumens and GA was present in supernatants of KO null ileal washings and colonic homogenates. These results suggests that Paneth cells play a novel but major role in mouse starch digestion by secreting GA in a spliced isoform, MGAs, into the ileal lumen that is preserved in the lumen of the colon.

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Gastric Acid Neutralization and Protease Inhibition Improves Peroral Recombinant Aden~Associated Virus-Mediated Gene Delivery to the Intestines Guohong Shao, Kristin C. Baekstrom, Qin Huang, Thomas J. Sferra

Gene transfer to the gastrointestinal tract mediated by recombinant adeno-associated virus (rAAV) vectors has many potential applications including complementation of single gene disorders, genetic immunization, and production of therapeutic molecules to act at local or distant sites. In vitro studies indicate that rAAV serotype 2 vectors are adversely affected by conditions within the GI tract (e.g. low intragastric pH, secreted proteases). The objective of this study was to evaluate whether acid neutralization and protease inhibition can improve rAAV gene delivery to the intestines. Methods: An rAAV type 2 vector (10 ~~ infectious units) carrying a [3-galactosidase expression cassette was administered with or without sodium bicarbonate (NaHCO3; 1% solution) and aprotinin (protease inhibitor; 3 trypsin inhibitory units) to fasted, adult, FVB/N mice via a feeding tube. Groups of mice were sacrificed at 1.5 hrs and 2 wks post-vector administration. The small intestines were harvested and divided into thirds. Lumenal contents of each segment were evaluated for the presence of functional rAAV by measuring the ability to transduce HeLa cells (e.g. mediate [3-galactosidase expression). Intestinal tissues were evaluated for gene transfer by: (i) a PCR assay for vector genomes and (ii) histochemical staining for [3-galactosidase activity. Results: At 1.5 hrs post- rAAV administration, mice (n = 2) receiving rAAV plus NaHCO3/aprotinin had greater amounts of functional rAAV within the lumen of the small intestine than mice receiving rAAV alone (n = 2). The [3-galactosidase level following transduction of HeLa cells for all segments in the NaHCO3/aprotinin group was 277 ( +/-216 S.D.) times (X) the [3-galactosi- dase level in control, nontransduced HeLa cells and the rAAV-alone group was 9X (+A16 S.D.) controls. The distal intestinal segment of each mouse contained 58 and 176X control levels in the NaHCO3/aprotinin group and 1.0 and 1.2X controls in the rAAV-alone group. At 2 wks post-vector administration, vector genomes were present only within the intestinal tissue of mice receiving the NaHCO3/aprutinin. [3-galactosidase positive cells were not present in either group of mice. Conclusions: Peroral delivery of rAAV type 2 vectors to the intestines can be enhanced by neutralization of gastric acid and inhibition of gastrointestinal proteases. Further studies are necessary to determine whether increases in transgene expression can be achieved.

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Cathepsin L Expression Is Modulated by GATA-4 and Cdx2 During Intestinal Epithelial Cell Differentiation Richard K. Kim, Joao P. Teixeira, Eun Ran Suh, Peter G. Traber, Francois Boudreau

INTRODUCTION: Intestinal epithelial cell differentiation is thought to be orchestrated by the combinatory action of several transcription factors. We have preciously shown that conditional expression of Cdx2 in undifferentiated intestinal epithelial cells regulates prolifer- ation and differentiation. Moreover, we have recently reported that transcriptional activation of Sucrase-lsomaltase, an enterocyte gene specifically induced during differentiation, is modulated by the synergistic interaction of HNF- la , Cdx2 and GATA-4 transcription factors. AIM: To investigate the combined role of Cdx2 and GATA-4 on gene expression during intestinal epithelial cell differentiation. METHODS AND RESULTS: The profile of GATA-4 expression was first evaluated by Western during the course of IEC-6/Cdx2 differentiation. GATA-4 protein expression was strongly induced following the activation of Cdx2 expression. A microarray analysis of genes differentially expressed during IEC-6/Cdx2 differentiation identified Cathepsin L as a putative induced target of GATA-4 and Cdx2. The expression of catalytic Cathepsin L was induced during IEC-6/Cdx2 differentiation as confirmed by

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