Characterization of the rabbit gastric epithelial lineage progenitors in short-term culture

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1 Cell Tissue Res (2001) 306:65 74 DOI /s REGULAR ARTICLE S. M. Karam G. Alexander V. Farook A. Wagdi Characterization of the rabbit gastric epithelial lineage progenitors in short-term culture Received: 13 February 2001 / Accepted: 11 May 2001 / Published online: 19 July 2001 Springer-Verlag 2001 Abstract Little is known about the mechanisms that establish and maintain the proliferation and differentiation programs of the gastric epithelium. This is largely due to the complexity of the gastric epithelial units and the presence of the different epithelial lineage progenitors among heterogeneous populations of various mature cell types. This study is undertaken to establish an in vitro system highly enriched for gastric epithelial lineage progenitors. By using adult male rabbits, a simple method of isolating gastric epithelial cell fractions enriched in lineage progenitors was applied. Cultured cells labeled with bromodeoxyuridine were characterized by using lectin and immunohistochemical studies at light- and electronmicroscopical levels. Analysis of primary cultures derived from the progenitor cell region of the epithelial units revealed that this system can support the proliferation and some of the differentiation programs of the progenitor cells. Cultured cells can be maintained for up to 5 days, while retaining most of the morphological features, molecular markers, and dynamic behavior of gastric epithelial progenitors. Differential cell counts at 1-day culture revealed that, while the undifferentiated progenitors formed about 30% of all attached cells, prepit, pit, and preparietal cells represented about 30%, 10%, and 2%, respectively. By 3 days, the increase in the percentage of pit and preparietal cells up to 25% and 9%, respectively, reflected their production in vitro. In conclusion, we have established a culture system enriched for gastric epithelial lineage progenitors that would hopefully allow the identification of factors and mechanisms involved in controlling their proliferative activity and differentiation pathways. This study is supported by Kuwait University grant MA 038 to S.M.K. S.M. Karam ( ) G. Alexander V. Farook A. Wagdi Department of Human Anatomy, Faculty of Medicine & Health Sciences, United Arab Emirates University, PO Box 17666, Al-Ain, United Arab Emirates ekaram@uaeu.ac.ae Fax: Keywords Gastric mucosa Cell isolation Cell proliferation Stem cell Gastric gland Epithelial progenitor Rabbit (New Zealand) Introduction The epithelial lining of the oxyntic mucosa of the rabbit stomach forms short pits continuous with long tubular glands made of isthmus, neck, and base regions (Fig. 1). There has been a general agreement that cell proliferation in the gastric glands occurs in the isthmus region (Plenk 1932; Stevens and Leblond 1953), where cells quickly acquire [ 3 H]thymidine labeling after injection (Messier and Leblond 1960; MacDonald et al. 1964; Kataoka et al. 1989). This proliferative cell region has been found recently to include various types of epithelial lineage progenitors in mice (Karam and Leblond 1992), rabbits (Karam and Forte 1994), and humans (S.M. Karam, T. Straiton, W. Hassan, C.P. Leblond, unpublished observation). By tracing the ultrastructural changes and [ 3 H]thymidine labeling pattern of these progenitors, it was possible to define the gastric epithelial lineage pathways in the pit-gland units of the mouse stomach (Karam 1993; Karam and Leblond 1993a, 1993b, 1993c, 1993d). The undifferentiated progenitor (also called granule-free cell) acts as the pluripotent stem cell of the epithelium that gives rise to three different committed progenitors of the three major cell types: parietal (or oxyntic), zymogenic (chief), and pit (surface mucous) cells. The differentiation of these cells occurs during a spacially well organized bipolar cell migration from the isthmus to the surface epithelium and to the blind end of the gland (Karam 1993; Karam and Leblond 1993a, 1993b, 1993c, 1993d). One central question in gastric epithelial cell biology is the identification of factors that control proliferation and differentiation of the gastric epithelial progenitors. Resolution of this question has been difficult due to the lack of an in vitro functioning system which includes these progenitors. The use of gastric epithelial cell lines

2 66 gradients of intestinal epithelial cells was applied, with some modifications to isolate sequential cell fractions from the gastric epithelium along the pit-isthmus axis. The experimental procedure was approved by the Medical Research Committee of Kuwait University. The stomach lumen was washed with physiologically buffered saline (PBS) and clamped at the pylorus-corpus junction to exclude the pyloric antrum. Then the stomach was filled through the esophageal opening with Eagle s basic medium (BME) containing hyaluronidase (Sigma, St. Louis, Mo.; concentration 1.5 mg/ml) and incubated for 10 min in a shaking water bath at 37 C. The solution was collected from the stomach lumen and discarded. To allow cellular release, 30 ml PBS containing 2 mm ethylenediaminetetraacetic acid (EDTA) was introduced into the stomach and gently shaken on an ice bag for 1 min. The luminal contents were collected in a sterile tube, which was marked as fraction 1. The previous step was repeated to obtain nine additional cell fractions. All cell fractions were spun down at 100g for 5 min. The supernatant was discarded and the cells were resuspended in 30 ml of BME. To verify the presence of cells in the various fractions, a drop of each fraction was examined under the differential interference (Nomarski) optics of the AH3 Olympus microscope (Olympus Optics, Tokyo, Japan). To confirm the extent of cell release after different fractions were obtained, the remaining stomach tissue was processed for plastic embedding and toluidine blue staining (Karam and Leblond 1992). Short-term culture Fig. 1 Representation of the pit-gland unit of the oxyntic mucosa of the rabbit stomach. The pit and the luminal surface seen at the top are lined by mucus-secreting pit cells. According to the distribution of cell types, the gland is divided into three regions: isthmus, neck, and base. While the acid-secreting parietal cells are scattered in the three regions, progenitor cells are found in the isthmus, neck cells in the neck, and zymogenic cells in the base (Motoyama et al. 1986) established from adenocarcinomas have not provided an ex vivo model of the diversified gastric epithelial progenitors. In addition, these transformed cells may not respond to factors that affect signaling pathways of cell proliferation and differentiation in vivo. In this study, we isolated sequential fractions of gastric epithelial cells along the pit-isthmus axis of the rabbit stomach and established a primary culture system enriched in the undifferentiated and committed progenitors. Then, based on various microscopic techniques, we characterized the in vitro features of these progenitors. Materials and methods Cell isolation Male New Zealand rabbits (Animal Resources Center, Kuwait University) weighting 2 3 kg were anesthetized and their stomachs were removed. The method of Weiser (1973) for isolating The cell fractions were centrifuged for 4 min and each of the cellular pellets was resuspended in 6 ml Iscove s modified Dulbecco s medium (IMDM; Gibco, Grand Island, N.Y.) containing 5% fetal calf serum, penicillin (Sigma; 100 U/ml) and streptomycin (Sigma; 100 µg/ml). The suspended cells ( cells in 0.5 ml medium) were seeded in 24-well plate (Nunc, Naperville, Ill.) or 8-well Lab Tek chamber slides (Nunc). In some cases, the multiwell tissue culture plates were precoated with extracted rat tail collagen type I (Elsdale and Bard 1972). Cells were allowed to attach and grow at 37 C in a humidified atmosphere containing 5% CO 2 /95% air. After 1 day, the medium with unattached cells was discarded and fresh medium was added. The medium was replaced after 2 more days. For counting, cells were detached by trypsin- EDTA (Sigma) treatment for 10 min, and counted in triplicate using a hemocytometer. The mean ± SE of three different experiments was calculated. Immunohistochemistry and lectin histochemistry Cells were fixed in Bouin s solution and subsequently permeabilized in PBS containing 0.1% Triton X-100. After blocking in 0.1% bovine serum albumin (BSA) in PBS, cells were incubated with either primary antibodies or lectins. Monoclonal rabbit anti-alpha (kind gift from Dr. Adam Smolka; Smolka and Swiger 1992) or anti-beta (kind gift from Drs. Dar Chow and John Forte; Chow and Forte 1993) subunits of the H,K-ATPase specific for parietal cells were diluted in PBS containing 0.05% Tween-20 and 0.1% BSA. To visualize antigen-antibody binding sites, secondary anti-mouse IgG (Sigma) conjugated with fluorescein isothiocyanate (FITC) was added to the cells at 1:200 dilution for 1 h at room temperature. FITC-labeled Ulex europaeus agglutinine I (UEA I; Sigma) and Griffonia simplifolica II (GSII; Sigma) lectins were respectively used as markers for the mucus-secreting pit and neck cells (and their committed progenitors). Following 1 h lectin incubation, the cells were washed with PBS. All probed cultured cells were mounted with coverslips using PBS/glycerol (1:1). Cells were examined under the fluorescent optics of the Olympus microscope. Electron microscopy In some experiments, cells grown in multi-well plates were fixed for 20 min in sodium cacodylate buffer containing formaldehyde

3 mix (Karam and Leblond 1992). After several buffer washes, the cells were postfixed for 10 min in reduced osmium tetroxide, dehydrated, and then infiltrated/embedded in Araldite blocks. Thin sections were cut, stained with uranyl-lead and examined under the Jeol 1200 EM (Jeol Co, Tokyo, Japan). For antibody- and lectin-binding analysis at the level of the electron microscope, cultured cells were fixed in 0.1 M sodium cacodylate buffer containing 4% paraformaldehyde. The cells were then infiltrated and embedded in LR white resin (Sigma). Semithin and thin sections were cut and placed respectively on slides and nickel grids. For antibody or lectin probing, sections were first blocked for 45 min in PBS containing 0.5% BSA and 0.05% Tween-20. Some sections were then incubated with blocking solution containing the primary antibody (anti-h,k-atpase) for 2 h. After several washes in PBS, the antigen-antibody sites were labeled with gold during 45 min incubation in rat anti-mouse IgG conjugated with 18-nm gold particles (Sigma). For lectin probing, sections were incubated with 10 nm gold-labeled UEA-I or GSII lectins (Sigma). All sections were washed in PBS, then immersed in distilled water and allowed to dry. After uranyl-lead staining, probed sections were examined under the EM. Measurement of the proliferative activity of cultured cells To quantitate proliferation of gastric epithelial progenitors in primary cultures, cells were grown in IMDM medium containing 5% fetal calf serum. After 1 day, the medium was replaced with IMDM containing insulin (5 µg/ml) or hepatocyte growth factor (HGF; 50 pm). Cells were incubated at 37 C for 2 more days and then bromodeoxyuridine (BrdU; Sigma; 50 mm) was added 6 h before fixation in Bouin s solution. The cells were then washed in PBS and immunostained using goat anti-brdu serum (kind gift from Dr. Jeff Gordon; Cohn and Lieberman 1984). The effect of the insulin or HGF on BrdU labeling was quantitated by examining at least 300 nuclei of epithelial cells. Results Cell isolation and appearance of cell fractions Light-microscopic examination of the gastric mucosa after the procedure of cell isolation showed that only the cells lining the luminal portion of the epithelial units were released, whereas deep regions of the gastric glands which included mucous neck, parietal, and zymogenic cells remained intact (Fig. 2). Under the phase-contrast microscope, the isolated cell fractions appeared to include mainly dispersed cells, in addition to some cellular clumps (not shown). The clumps tended to be more in fractions 1 5, which included cells rich in mucous granules. Fractions 8 10 were more dispersed and were expected to include cells from the isthmus region (poorly differentiated cells with little or no mucous granules). Therefore, cells tended to release in a pit-to-isthmus direction. Primary culture system enriched in the gastric epithelial progenitors Plating of the various isolated cell fractions resulted in the confirmation of fractions 8 10 being rich in highly proliferative, probably poorly differentiated (progenitor) cells. After 1-day culture, while most of the cells in initial fractions were floating in the medium, in fractions 8 10, small groups of tightly packed adherent cells were attached to the culture dish (Fig. 3A). After 3 days, the attached cell groups became larger and fused with each other to form a monolayer. By 5 days, some cells started to detach, while cytoplasmic vacuoles appeared in some other cells. Cell counts carried out with the hemocytometer after trypsin-edta incubation revealed that in 1-day cultures there were cells/well, whereas by 3 days, there were an average of cells/well. Analysis of the ex vivo features of the gastric epithelial lineage progenitors Lectin-histochemical and immunohistochemical analysis Lectins known as markers of surface mucous (pit) cells and mucous neck cells in the mouse stomach (Falk et al. 1994) were initially used to probe paraffin sections of normal rabbit gastric mucosa. It was found that, in the rabbit stomach, UEA I and GSII lectins respectively bind to pit and neck cells (not shown). Therefore, these two lectins in addition to antibodies specific to the alpha- and beta-subunits of the H,K-ATPase were used to identify cells in culture. Probing of cells cultured for 1 day and 3 days with UEA I lectin showed that many of the attached cells belonged to the pit cell lineage (Fig. 3A C). Many of these cells were produced in culture, as demonstrated by the BrdU labeling (red nuclei in Fig. 3A, B). However, few H,K-ATPase-labeled cells belonging to the parietal cell lineage were found in 1- and 3-day cultures and did not incorporate BrdU (Fig. 3D). Similarly GSII labeling demonstrated that a small number of cells in the 3-day culture belonged to the neck cell lineage (Fig. 3E). The nuclei of most of these GSII-labeled cells did not incorporate BrdU; thus they were not produced in vitro. When 3-day cultured cells were processed for LR-white embedding and semithin sectioning, the UEA I lectin labeling was found to be confined to the apical side of the cells (Fig. 3C). The GSII lectin appeared to label a small round area in the cytoplasm of a few cells (not shown). Electron-microscopic analysis 67 Cell types present in cultured fractions 8 10 were identified based on previously reported criteria (Karam and Leblond 1992; Karam and Forte 1994). In addition to the few fully differentiated mucous, parietal, or enteroendocrine cells observed, many small cells predominated in the 1- and 3-day cultures (Fig. 4). The nucleus in these cells tended to include much diffuse chromatin and prominent nucleoli. The cytoplasm exhibited signs of poor differentiation: many free ribosomes, few small mitochondria, and small Golgi stacks showing little or no signs of secretory activity. But there were sufficient vari-

4 68 Fig. 2A, B Light micrographs of toluidine blue-stained semithin sections of rabbit gastric mucosa. A Intact mucosa. Surface mucous or pit cells (s) form the lining of the luminal surface and the pit walls. Mucous neck cells (n) are seen deep in the glands, and some zymogenic cells (z), at the bottom. The area between pit and neck cells contains poorly differentiated progenitor cells with little or no secretory granules (arrow). Large parietal cells (p) are scattered among neck and zymogenic cells. B Mucosa after cell isolation procedure. While the lining epithelial pit and progenitor cells are removed, the connective tissue elements including capillaries (c) are intact. Note the presence of parietal (p), neck (n) and zymogenic (z) cells. Bar 40 µm Fig. 3A E Single and double labeling of cultured gastric epithelial cells. A One-day cultured cells incubated in the presence of bromodeoxyuridine (BrdU) to label cells produced in vitro. The cells were probed with anti-brdu (red) and then by Ulex europaeus agglutinine I (UEA-I) lectin (green). Note that some of the newly produced cells express the lectin specific for pit cell lineage. B Cells after 3-day culture probed as in Fig. 3. Note the increase in the number of BrdU-labeled cells belonging to the pit cell lineage. C Semithin section of 3-day cultured cells processed in LR-white resin and probed with FITC-labeled UEA-I lectin. Some cells exhibit apical staining, others are negative. D Cells after 3-day culture probed with anti-brdu and then with anti-beta subunit of the H,K-ATPase. Only one H,K-ATPase-labeled cell (green) is seen at the right side. All BrdU-labeled cells do not express H,K-ATPase. E Cells after 3-day culture probed with anti- BrdU and then with neck cell-specific Griffonia simplifolica II (GSII) lectin. Note that GSII labels small round areas in the cytoplasm of neck cells. These areas probably represent the accumulated secretory granules. Very few of the BrdU labeling colocalizes with GSII. Bar 20 µm

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7 Fig. 5 Counts of various progenitor and mature cell types identified under the electron microscope in 1- and 3-day cultures of gastric mucosal fraction number 9. For each of three different experiments, about 100 cells were scored and categorized to a specific progenitor or mature cell type. The mean ± SE are plotted Fig. 4A D Electron micrographs of gastric epithelial progenitors as they appear in 3-day culture of fraction 9 (N nucleus). A Undifferentiated (granule-free) progenitor attached to neighboring cells at right and left by junctional complexes (J), a feature of epithelial cells. The apical membrane exhibits few short microvilli (arrows) covered with relatively dense glycocalyx. At the two poles of the nucleus the Golgi stacks (G) appear very small with no signs of secretory granule production. Note the dense granules of prepit cell at the left (m mitochondria). B Prepit cell exhibiting few dense secretory granules at the apex. The apical membrane projects glycocalyx-coated microvilli which appear more numerous than those of the undifferentiated cell in A. C Preparietal cell characterized by numerous elements of tubulovesicles (large arrows) next to the Golgi apparatus (G) in the perinuclear cytoplasm. The mitochondria (m) are relatively numerous compared with other progenitors. At the upper right, the apical membrane projects microvilli (small arrows) with no apparent glycocalyx. D Preneck cell characterized by few pale secretory granules at the left side of the nucleus. Some of the granules (arrows) exhibit small dense core characteristic of neck cell lineage; others appear altered (very pale and include small vesicles). At the lower right corner is a small portion of an enteroendocrine cell (ec) carrying numerous small dense secretory granules. Bar 1,000 nm ations among these cells to identify four different types. The first cell type was characterized by having no secretory granules and no tubulovesicles. Also, the Golgi apparatus was very small to justify placing these cells in the distinct category of undifferentiated (granule-free) progenitor cells (Fig. 4A). These cells were numerous at both 1- and 3-day cultures, 29.1% and 31.2%. The second type of progenitor cells had a few dense secretory granules at the cell apex (Fig. 4B) which were more or less ovoid and very similar to the secretory granules of pit cells, and therefore these cells are the prepit cells. They were relatively numerous in both 1- and 3-day cultures, where they represented respectively 30.6% and 21.3%. The third type was characterized by apical microvilli without any apparent glycocalyx. In the cytoplasm, the main feature was the presence of numerous small tubules and vesicles (Fig. 4C) that probably represent the H,K-ATPase-containing tubulovesicular system of parietal cell lineage. These features justify considering these cells as the preparietal cells. They were rare (1.6% of total cells) in 1-day cultures, but increased to about 8.5% after 3 days. The fourth cell type displayed a few cytoplasmic granules, which usually accumulate at one side of the nucleus (Fig. 4D). Some of these granules appeared to have moderately dense contents with very dark peripheral core similar to those of neck cell lineage (Karam and Forte 1994). Thus, these cells are the preneck cells that represented about 14.2% in 1-day cultures and 7.1% after 3 days. The mature cells observed in the EM were relatively few. While the percentage of enteroendocrine, parietal, and neck cells decreased from 1- to 3-day cultures, counts of pit cells showed an increase from 10.7 to 25.4% (Fig. 5). For immuno- and lectin-cytochemical analysis at the level of the electron microscope, members of UEA I-labeled pit and GSII-labeled neck cell lineages were identified by the presence of gold particles on the secretory granules and apical membranes of the cells (UEA-Ilabeled prepit cells are seen in Fig. 6). Therefore, the presence of four types of lineage progenitors in the culture system was confirmed by using different microscopy techniques. Effects of insulin and hepatocyte growth factor Incubation of the cultured cells in the presence of insulin or HGF showed that both factors have mitogenic effects on the gastric epithelial progenitors (Fig. 7A). The percentage increase in BrdU labeling was calculated and found to be about 114 and 143 for HGF and insulin, respectively. The response to HGF was also tested at different concentrations of pm. The observed data demonstrated a dose-dependent increase in the mitogenic response to HGF (Fig. 7B). At concentrations of 10 pm, 25 pm, 50 pm, and 100 pm, the percentage increase in BrdU labeling was, respectively, 61, 122, 167, and 228. Discussion Most of what is known about epithelial cell renewal in the gastric glands has come from studies conducted in vivo, primarily on mice, and has been recently expanded by the use of genetically manipulated mice (reviewed by Karam, 1995, 1998). The early events that occur during gastric epithelial cell renewal include proliferation of undifferentiated (granule-free) progenitor cells and production of committed progenitors. Little is known about these events due to lack of an ex vivo system which reproduces the proliferation program of the gastric epithelial progenitors. The development of a primary culture system that would allow studies on the gastric epithelial progenitors 71

8 72 Fig. 6A, B Immunogold localization of UEA-I lectin in ultrathin sections of LR-white-embedded cells of fraction 9 after 3-day culture. A Prepit cell identified by the presence of gold particles (arrows) in the apical secretory granules which appear pale after processing in LR white. B A close-up of two different progenitor cells separated by a large horizontal arrow. The cell above the arrow is defined as preparietal cell due to relatively large mitochondria (m). The cell below the arrow is defined as prepit cell due to presence of small dense gold particles (small arrows) of UEA-I lectin on its apical membrane. Bar 500 nm Fig. 7A, B Effects of insulin and hepatocyte growth factor (HGF) on cellular proliferation in primary cultures enriched in rabbit gastric epithelial progenitors. The results shown are the mean of three experiments, each run in triplicate. Data are shown as the percentage ± SE. A The effects of insulin and HGF. B The effects of increasing doses of HGF is considered a worthwhile objective, because it has recently been proposed that gastric cancer, the second leading cause of cancer deaths worldwide (Parkin 1998), originates in epithelial progenitors (Correa 1992) and there is evidence that a carcinogenic event taking place within such progenitors is a common cause of cancers (Del Buono and Wright 1995). Since biologically produced carcinogens can induce gastric cancer (Correa 1992), such carcinogens are likely to be targeted to these progenitors. Therefore, to understand the pathogenesis of gastric cancer, it is necessary to improve our knowledge of these lineage progenitors. Several different methods based on Percoll density gradients (Mardh et al. 1984) or centrifugal elutriation are described in the literature to obtain fully differentiated gastric epithelial cells enriched in parietal (Chew 1994), mucous (Boland et al. 1990), or pepsinogen-producing cells (Chen et al. 1991). In studies aimed at establishing new primary culture systems from fetal rat (Tommeras et al. 1997) or human (Basque et al. 1999) stomachs, emphasis has been placed on the developing mature parietal or chief cells. No previous attempts have been made for the isolation and in vitro characterization of gastric epithelial progenitors of adult stomachs. In order to carry out experiments aimed at elucidating the factors that may alter these epithelial progenitors in adults and to study the role they play in the production of the main cell lineages, a simple method of isolating cell

9 fractions enriched in undifferentiated and committed progenitors from the rabbit gastric mucosa and maintaining them in short-term culture system has been described in the present study. Since the gastric epithelial cells are organized in anatomically well-characterized units (Fig. 1) and it has been established that the units progenitors are anchored in the isthmus region (Karam and Leblond 1993a), the approach of Weiser (1973) to isolate successive fractions of the intestinal epithelium will be applicable to the rabbit stomach for obtaining epithelial cell fractions enriched in the lineage progenitors. Our short-term culture of the gastric epithelial progenitors (fractions 8 10) allowed exclusion of all zymogenic cells (located at the base region of the glands), most mucus-secreting pit and neck cells (located respectively in the pit and neck regions), most parietal and enteroendocrine cells (scattered throughout the epithelial units), as well as the connective tissue elements, which remained in the mucosa after cell release (Fig. 2B). Moreover, cell counts under the electron microscope demonstrate that the percentage of neck, parietal, and enteroendocrine cells at 1-day culture is minimal (Fig. 5), whereas undifferentiated and committed progenitors are numerous, with the exception of preparietal cells, which are also rare in vivo (Karam 1993; Karam and Forte 1994). In this study, our emphasis has been on the use of a number of different approaches to characterize the isolated cell fractions which are maintained in culture for up to 5 days. To examine the cells in detail, we have chosen two time points: 1-day and 3-day cultures. Morphologically, the cells are clearly epithelial as demonstrated from their appearance under the electron microscope which revealed their junctional complexes, microvilli, and polarity (Fig. 4). Moreover, specific features characteristic of gastric epithelial lineage progenitors are also demonstrated (Fig. 4). Using lectin and immunofluorescence or gold techniques, the previously characterized molecular markers appear to be preserved in these epithelial progenitors (Figs. 3, 6). Further characterization of the ex vivo features of the gastric epithelial lineage progenitors has been directed toward the demonstration of their proliferative activity. Incubation of the 1-day cultured cells with BrdU for 6 h before their fixation revealed about 20% labeled nuclei (Fig. 3A). In an attempt to modulate this proliferative activity, cultured progenitors were labeled with BrdU in the presence of insulin or HGF, known for their mitogenic effects on the gastric epithelium using different signaling pathways (reviewed in Podolsky 1994). While the effect of insulin is known to gut cell biologists and it has become a common factor added to culture systems to enhance cell proliferation, few data are available regarding the effects of HGF (Kong et al. 1998). Therefore, we first showed that cultured epithelial progenitors of adult rabbit stomach respond to insulin and then demonstrated the mitogenic effect of HGF. By comparing the percentage of cells found in 1-day and 3-day cultures, it appears that our primary culture system supports the differentiation program of pit cell lineage, where by 3 days the percentage of pit cells is doubled. Also, this culture system supports the early differentiation program of parietal cell lineage, where the percentage of the progenitors of parietal cells is increased by 3 days. In conclusion, the short-term culture of gastric mucosal cell fractions represents an in vitro functional assay system that can be used for defining factors and/or compounds that influence proliferation of gastric epithelial progenitors. In addition, this assay system could make it possible to begin to dissect the molecular mechanisms underlying the remarkable events that occur during renewal of the gastric epithelium. Acknowledgements The authors thank Drs. Gerald Buzzell and Charles Leblond for their critical comments on the manuscript. The authors would like to thank Drs: John Forte, Gregory Aponte, and Franky Chan for their support and valuable discussion at the beginning of this study. 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