The lymphoepithelial phenomenon of the stomach. A case report

A large number of lymphocytes each surrounded by a clear halo were found in the foveolar epithelium of the fundic region and the antrum in a gastric biopsy from a patient with epigastric complaints. Electron microscopical studies demonstrated the presence of intraepithelial "in-halo" lymphocytes both underneath, between two contiguous, and within foveolar gastric cells. Normal numbers of lymphocytes (without haloes) were found in the lamina propria, suggesting that this epithelial lymphocytosis may be unrelated to "classical" chronic gastritis. The possibility of an autoimmune phenomenon at the foveolar cell level was discussed. The absence of intraepithelial lymphocytes in the glandular epithelium suggests that this phenomenon may be unrelated to the type of autoimmunity found in pernicious anaemia.     

Telomere length variations in 6 mucosal cell types of gastric tissue observed using a novel quantitative fluorescence in situ hybridization method

We developed a novel method for evaluating telomere length in 6 cell types of noncancerous and cancerous mucosal tissues from 11 cases of gastric neoplasm using the quantitative fluorescence in situ hybridization method with telomere and centromere peptide nucleic acid probes. Our telomere length estimates were determined from the background-corrected telomere intensity divided by the background-corrected centromere intensity (telomere-to-centromere ratio). Our results indicated that telomere lengths in each of the cases studied were reduced in turn from fibroblasts to fundic gland cells, to glandular neck cells, and then to surface foveolar cells. However, the telomere lengths of intestinalized cells located among fundic glands were not always shorter than those of the other cell types, as reported previously by others. Helicobacter pylori infection was suggested to induce the telomere shortening seen in the fundic glands. Although the mean telomere lengths varied among the 8 gastric cancer cases, correlation of the telomere lengths with the Ki-67 labeling index was established after normalization with the fibroblast measurements. We conclude that our telomere-to-centromere ratio method can reliably estimate the telomere lengths of the 6 cell types in the gastric mucosa and clarifies the relationship between proliferative activity and the telomere length of cancer cells.     

Gastric adenomas and superficial adenocarcinomas display distinct patterns of mucin carbohydrate and core protein expression

AIMS: To investigate the histogenetic relationship between gastric epithelial neoplasms we studied differences in expression of mucin carbohydrate antigens and mucin core protein, in normal and metaplastic gastric mucosa, and in gastric adenomas and superficial adenocarcinomas. METHODS AND RESULTS: We generated four monoclonal antibodies, including HGM72/75 against human gastric mucin and HCM14/21 against human colonic mucin, and investigated immunoreactivities of these antibodies and MUC2 protein expression in normal and metaplastic gastric mucosa, adenomas (15 samples) and superficial adenocarcinomas (intestinal-type, 77; diffuse-type, 59 samples). HGM72 reacted with mucous neck cells of the fundic glands and with pyloric glandular cells whereas HGM75 stained foveolar cells and metaplastic goblet cells. Weak binding of HCM14/21 and strong staining with MUC2 were found in metaplastic goblet cells. Binding of HGM75, HCM14, MUC2, but not HGM72 was high in adenomas. An equivalent staining with HGM72 and HGM75 with low expression of MUC2 and HCM14 was shown in intestinal-type carcinomas while the diffuse-type demonstrated more strong reactivity with HGM75 than with HGM72, MUC2 and HCM14. Little binding of HCM21 was observed in any specimens. CONCLUSIONS: This study demonstrates that adenomas predominantly have a intestinal phenotype, but both types of adenocarcinomas retain some cells with a gastric phenotype during the early steps of neoplastic development.     

Repair of gastric ulcer

Summary By a cryosurgical method a mucosal defect was produced in the body of the rat stomach, and repair of the gastric ulcer was studied with ³H-thymidine autoradiography. From 18 to 24 hours after cryoinjury, cell proliferation in the fundic mucosa was much increased, as indicated by an increase in the labeling index of the cells in the proliferating cell zone of the mucosa, or by reactive incorporation of ³H-thymidine into the mucous neck, chief and parietal cells. Spatially, the increased cell proliferation was found in a region of the mucosa more than 600 rn distant from the injury, and lasted for more than 14 days. The mucous neck and chief cells around the ulcer which had incorporated ³H-thymidine seemed to transform into flat cells after mitotic division and then continued to divide. After 3–5 days, so called regenerating epithelium or covering epithelium appeared around the ulcer. The upper part of this regenerating epithelium consists of tall columnar cells, the lower part is composed of cystic glandular structures, in which many ³H-thymidine incorporating cells were seen. The formation of new glands in the mucosal defect appeared to take place by budding from these cystic glandular structures with subsequent differentiation of surface epithelial cells. After 3 weeks the mucosal defect was covered by mucinous glandular structures similar to the proper pyloric mucosa. The proliferating cells were confined to the middle level in the regenerated mucosa.Dedicated to Professor Dr. W. Maurer on the occasion of his 75^th birthdayThis work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, He 537/5Scholar of the Alexander von Humboldt-Foundation     

Pathomorphological and biosynthetic changes in gastric epithelium during chronic duodenal ulcer

We studied structural changes and biosynthetic processes in the gastric epithelium in patients with chronic duodenal ulcer. The severity of alterative ultrastructural changes in surface epithelial and glandular cells progressively increased from proximal to distal portion of the stomach. Biosynthesis of nucleic acids within the epithelium depended on the morphofunctional state of epitheliocytes. DNA biosynthesis in the fundus was less intensive. The intensity of RNA synthesis did not differ between various gastric regions. Our results suggest that structural and metabolic dimorphism of the fundic and pyloric epithelium underlies pathological changes in the mucosa during ulcer disease.     

HYPERPLASTIC FUNDIC GLAND POLYP OF THE STOMACH

A case of a solitary pedunculated gastric polyp occurring In the fundic mucosa of a 47-year-old woman is reported because of its unique morphology. The polyp showed a peculiar lobulated configuration supported by smooth muscle bundles, and its epithelial component was composed mainly of a proliferation of relatively normal but disorganized fundic glands containing variable numbers of mucous, parietal, argyrophile, and chief cells. Although these histologic features may suggest a hamartoma-like quality, the lesion probably represents a peculiar form of localized hyperplastic process of the gastric mucosa exhibiting fundic glandular cell differentiation.     

Cell proliferation and differentiation in intramucosal and advanced signet ring cell carcinomas of the human stomach

Summary In order to study the progression of signet ring cell carcinomas in the human stomach, we compared cell proliferation and differentiation between small and large intramucosal cancers, and between intramucosal and advanced cancers. Fine-structurally, signet ring cells were differentiated to 3 cell types: a foveolar, a glandular and an intestinal type. In the mucosa, the foveolar-type cells and glandular-type cells were distributed at the superficial and the deep zone, respectively. In the small mucosal cancers, intestinal-type cells were rare and a layered structure was often seen. In this structure, the mode of cell production resembled that in the normal gastric mucosa; the foveolar-type signet ring cells in the superficial layer were not proliferative and the proliferating cells were small cells in the middle layer and a few glandular-type cells in the deep layer. In the large mucosal and advanced cancers, intestinal-type cells and proliferating small round cells were often distributed throughout the depth of the mucosa, and signet ring cells of the foveolar type were also proliferative. These findings indicated that large part of the signet ring cell carcinomas initially form the layered structure and that it becomes indistinct while intestinal-type cells appear as the tumour grows. However, we found several small advanced cancers, lacking both the layered structure and the intestinal-type cells. These cancers appear to start without the layered structure and progress very rapidly.     

Solitary gastric polyps in the fundic gland area. A histochemical study

Six solitary gastric polyps in the acid-secreting fundic mucosa were histochemically investigated using the mucin histochemistry, immunoperoxidase method, and silver methods for endocrine cells. Histologically, the polyps were grouped into three types: they largely consisted of either hyperplastic foveolar cells (group 1), normal-appearing fundic gland cells with mild cystic changes (group 2) or hyperplastic fundic gland cells with cystic dilatation (group 3). The presence of parietal cells and mucous neck cells was confirmed in all polyps by the immunoperoxidase method using parietal cell autoantibody and the paradoxical Concanavalin A staining, respectively. Regarding the endocrine component, somatostatin-containing cells, Grimelius-positive argyrophil cells, and Fontana-Masson-positive enterochromaffin cells were scattered in the fundic gland area of the polyps as well as in the surrounding normal-appearing fundic mucosa. Gastrin-containing cells were absent. In one of the group 2 polyps and both group 3 polyps, a varying number of glicentin-containing cells were found among the fundic gland components: In one polyp in group 3, glucagon immunoreactivity was detected in the glicentin-containing cells. These findings suggest that some of the polyps express characteristics of the fetal fundic mucosa, since glicentin and glucagon immunoreactivities in normal human stomach have been detected exclusively in the fetal fundus.     

Lysozyme localization in normal and diseased human gastric and colonic mucosa. A correlative histochemical, immunohistochemical and immunoelectron microscopic investigation.

The distribution of lysozyme in normal and pathological human gastric and colonic mucosa was studied by light and electron microscopic immunocytochemical techniques and compared with histological and histochemical features. Lysozyme was localized in pyloric glandular epithelial cells, mucous neck cells of fundic glands, Paneth cells and some crypt cells of the mature colonic mucosa. In addition, lysozyme was detected in a large spectrum of "immature" or "regenerative" epithelium: neck cells of the gastric regenerative zone, undifferentiated columnar cells of surface and hyperplastic interfoveolar crests of the stomach, regenerative cells in a healed gastric ulcer, some goblet cells in incomplete intestinal metaplasia, cells of the regenerative zone at the bottom of colonic crypts and, finally, fetal intestinal epithelium. Electron microscopically, we localized lysozyme in the central core of mucous granules in the pyloric gastric glandular epithelium and in the dense mucous granules in gastric mucous neck cells. Lysozyme was also detected in some immature mucin-producing cells of the gastric regenerative zone and in the rough endoplasmic reticulum of surface hyperplastic columnar gastric cells. At the electron microscopic level, a peculiar correlation between the immunopattern of lysozyme and the morphology of mucous granules has been postulated. All our data support and extend the view that the presence of lysozyme may be related to cell immaturity as well as to a regenerative state of the cell. Finally, the lysozyme distribution and its relation to mucosubstances in gastric and colonic carcinoma suggest that lysozyme should not be considered an exclusive marker of cells of gastric derivation.     

Bidirectional gastric differentiation in cellular mucin phenotype (foveolar and pyloric) in serrated adenoma and hyperplastic polyp of the colorectum

This study examined whether gastric pyloric gland-type mucin is expressed in serrated adenoma (SA) and in hyperplastic polyp (HP) of the colorectum, and whether cellular position-based gastric differentiation is observed in these lesions as previously hypothesized. Immunostaining was performed for MUC6 and alpha-linked GlcNAc residue (pyloric gland-type mucin markers), human gastric mucin (HGM; foveolar-type mucin marker) and Ki-67 (proliferating cell marker) for 31 SA, 22 HP, 21 traditional tubular adenoma (TA) and 20 hyperplastic nodule (HN). MUC6 showed varying expression in SA, 22/31 (71.0%); HP, 15/22 (68.2%); TA, 2/21 (9.5%); and HN, 0/20 (0%) with significantly higher frequencies in SA and HP compared to those in TA and HN. The alpha-linked GlcNAc residue was found only in SA (3/31, 9.7%) and in HP (2/22, 9.1%). In SA and HP, HGM was typically expressed in the entire crypt length, but some reduction in expression was shown in the basal crypt portion below the proliferative zone. MUC6 and alpha-linked GlcNAc residues were expressed in the basal crypt portion below or below and including proliferative zone. These data demonstrate that SA and HP show bidirectional gastric (foveolar and pyloric gland) differentiation with respect to mucin cellular phenotype and the potential for cellular position-based differentiation, which mimics the gastric antral mucosa.     

Expression of integrin subunits correlates with differentiation of epithelial cell lineages in developing human gastric mucosa

Laminins may play important roles in gastric gland development due to the differential localization of their α chains in human fetal fundic (oxyntic) mucosa. To extend this hypothesis, the current investigation was undertaken to compare the anatomical and cellular distribution of epithelial integrin subunits with those of laminin α chains in the human stomach at different ages (8–22 weeks of gestation) using indirect immunofluorescence. In the body, fundus and antrum regions, the β1 and α6 subunits were associated with the entire epithelium at all developmental stages in the same way as laminin chains (α1/α5) detected with 4C7 monoclonal antibody. By contrast, the α3 and β4 subunits of α3β1 and α6β4 integrins together with the laminin α3-chain were concentrated in the surface and foveolus compartments composed of differentiating mucous cells. Most importantly, the α2 integrin subunit was expressed in a rather complex pattern: (1) it was located at the base and at cell-cell boundaries of surface/foveolar epithelium, (2) was specifically repressed in differentiating parietal cells, and (3) its expression increased in maturing glands, where it became concentrated at the basal pole of epithelial cells simultaneously exhibiting a strong reactivity for laminin-2 (α2-chain). Taken altogether, our observations provide new evidence for the implication of different laminins and their receptors in the development of all human gastric epithelial lineages, surface mucous cells or glandular cells. The coordinated expression of α2 and α3 integrin subunits as well as the cellular re-distribution of α2β1 integrin likely represent key events for the differentiation of glandular secretory cell types, especially maturing chief cells responsible for the synthesis/secretion of gastric digestive enzymes in fundic-type glands. Accepted: 26 March 2000     

Decreased gastric proliferation of foveolar epithelial cells after the eradication of Helicobacter pylori.

Increased epithelial cell proliferation is associated with an increased risk of gastric carcinoma. Helicobacter pylori infection is an established risk factor for gastric cancer and the organism has recently been classified as a group I carcinogen by an IARC working group. In this study, we describe differences in gastric epithelial cell proliferation between a H. pylori eradicated group (n = 21) and a not eradicated group (n = 8) after anti-H. pylori eradication therapy to show that increased cell proliferation is associated with H. pylori infection. H. pylori infection was determined by rapid urease test and immunohistochemical method with anti-H. pylori polyclonal antibody. Gastric epithelial cell proliferation was assessed using immunohistochemical method using Ki-67 monoclonal antibody. Ki-67 positive cells in H. pylori associated chronic active gastritis were observed in the glandular neck and the upper portion of foveolar epithelium. Patients who cleared their H. pylori infections showed a significant decrease of Ki-67 labeling index after therapy (0.73 +/- 0.10 vs. 0.48 +/- 0.08, p < 0.01). By contrast, Ki-67 labeling index before and after treatment in patients who remained positive for H. pylori showed no significant difference (0.78 +/- 0.08 vs 0.74 +/- 0.10, p > 0.05). These results indicate that H. pylori infection increases the proliferation of gastric foveolar epithelium, which is reduced by the eradication therapy. We suggest that anti-H. pylori eradication therapy can prevent mucosal cell proliferation to be closely associated with gastric carcinogenesis.     

IN SITU ANALYSIS OF TISSUE DYNAMICS AND p53 EXPRESSION IN HUMAN GASTRIC MUCOSA

In situ tissue dynamics were studied in 12 cases of human gastric mucosa, including normal gastric body mucosa and gastric glands with intestinal metaplasia, obtained from gastrectomy specimens of adenocarcinoma. Cell proliferation was determined by Ki67 immunoreactivity. DNA fragmentation was studied in situ by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In addition, p53 expression was examined by both immunohistochemistry and mRNA in situ hybridization. In the oxyntic gastric glands, Ki67 immunoreactivity was observed exclusively in the proliferative zone and TUNEL-positive cells were present predominantly in the surface foveolar epithelium. In the gastric glands with complete intestinal metaplasia, Ki67-positive cells were present in the lower portion of the glands and TUNEL-positive cells in the superficial epithelium. In the gastric glands with incomplete intestinal metaplasia, TUNEL-positive cells were detected in the lower gastric glands adjacent to cells immunoreactive for Ki67; the proportion of these gastric glands with TUNEL-positive cells (40 out of 108 glands) was significantly higher than for oxyntic glands (94 out of 620 glands) or for glands with complete metaplasia (31 out of 254 glands). Relatively strong p53 immunoreactivity and mRNA hybridization were also observed in the proliferative and apoptotic areas of gastric glands with incomplete intestinal metaplasia. These results indicate that incomplete intestinal metaplasia is associated with increased cell turnover and p53 overexpression, possibly in response to various noxious or DNA-damaging stimuli.     

Expression of extracellular matrix proteins in the fetal rat gastric mucosa

At gestational day 16 the epithelium of the rat stomach consists of a stratified layer of undifferentiated cells, and two days later glandular structures appear. The present study was carried out to identify extracellular matrix proteins that could be involved in the epithelial cell proliferation and differentiation processes that occur in the fetal rat stomach during this period. For comparative purposes the expression of the same components in the adult gastric mucosa was examined. Pregnant Sprague-Dawley rats received an intraperitoneal injection of 5-bromo-2’-deoxyuridine to label proliferating cells. One, 3.5, or 6 h post-injection the stomachs were excised and immediately frozen. The specimens were sectioned and stained with hematoxylin and eosin or for 5-bromo-2’-deoxyuridine, cytokeratin no. 8, H,K-ATPase, and the extracellular matrix proteins fibronectin, laminin, and collagens type I and IV. A stratified layer of proliferating cells was observed in the epithelium of the fetal stomachs, while in adult stomachs proliferating cells were detected in the isthmus/neck region of the glands. Cytokeratin, an epithelial cell marker, was sparse at gestational day 16 but abundant both at gestational day 18 and in the isthmus/neck region of gastric glands of the adult stomach. The parietal cell marker H,K-ATPase could not be detected in the fetal stomachs during this period. Fibronectin was observed in the stroma of both fetal and adult stomachs. Collagen type I could only be detected in the stroma close to the oesophagus at gestational day 16. Two days later, collagen type I was abundant in the lamina propria, the submucosa and in the serosa of the fetal stomachs. In adult tissue collagen type I was detected in the surface epithelium, the submucosa and in the serosa of the stomach. Collagen type IV and laminin were expressed in the lamina propria, the basement membranes around blood vessels, muscle cells, and nerve bundles, as well as in the serosa of both 16- and 18-day-old fetal and adult rat stomachs. In conclusion, a high cell proliferation rate was observed in the epithelium at both gestational days 16 and 18. The increased expression of cytokeratin observed during this period indicates that the epithelial character of the embryonic cells becomes more distinct, while the remarkable change in the expression of collagen type I might reflect an important role of collagen type I in the development of the gastric epithelium. Accepted: 2 September 1999     

Expression of Trefoil Peptides in The Gastric Mucosa of Transgenic Mice Overexpressing Transforming Growth Factor-α

Abstract

Overexpression of transforming growth factor-α (TGF-α) in the gastric mucosa of metallothionein-TGFα(MT-TGFα) transgenic mice leads to a marked alteration in the ontogeny of the fundic cellular lineages. Induction of the transgene leads to the over-production of mucous cells with a concomitant diminution in the development of parietal cell and chief cell lineages. We have sought to define more precisely the mucous cell lineages involved in the mucous cell hyperplasia in MT-TGFα mice by investigating the expression of trefoil peptides in MT-TGFα mice. MT-TGFα mice and their non-transgenic littermates were treated with cadmium sulfate beginning at 13 days of age. Animals were then sacrificed at intervals over the following 2 weeks and gastric mucosa was examined for expression of trefoil peptides and TGFα by immunohistochemistry and in situ hybridization. No TGFα mRNA expression could be demonstrated by in situ hybridization in non-transgenic mice. In MT-TGFα mice, in situ grains for TGFα mRNA were detected at the base of fundic glands in 13 day old animals, whereas the expression was observed more widely in the mucosa of older animals (28 days). TGFα immunoreactivity was observed in foveolar mucous cells and residual parietal cells in MT-TGFα mice at all ages. By in situ hybridization, pS2 mRNA was detected in the surface mucous cells in normal gastric mucosa. In MT-TGFα mice, pS2 mRNA was found throughout the expanded foveolar region. By in situ hybridization, spasmolytic peptide (SP) expression was observed in the region of the progenitor zone in both groups of mice. By immunohistochemistry, SP expression was noted in a broad band of mucous neck cells deep to the progenitor zone. No gastric expression of intestinal trefoil factor (ITF) was noted in either group of mice. The results demonstrate that the expansion of the foveolar mucous cell compartment in MT-TGFα mice is due to the hyperplasia of normal surface cells expressing their particular mucin-associated trefoil peptide, pS2.     

Association of spasmolytic polypeptide-expressing metaplasia with carcinogen administration and oxyntic atrophy in rats

Spasmolytic polypeptide (TFF2)-expressing metaplasia (SPEM) is a gastric metaplastic lineage associated with the development of intestinal-type gastric adenocarcinoma. To study the etiology of this potential neoplastic precursor metaplasia, we used surgical rat models of remnant gastric adenocarcinoma studied with and without exposure to nitroso carcinogen. Animals with truncal vagotomy without duodenogastric reflux procedures demonstrated normal mucous neck cell spasmolytic polypeptide (SP) immunostaining. In these animals, anti-proliferating cell nuclear antigen (PCNA)-labeled nuclei were found in the normal midgland progenitor zone. Rats that received anatomic alterations that augmented the degree of duodenogastric reflux, however, revealed expansion of basally placed SP immunoreactive cells with early phenotypic changes of SPEM. Seventy percent of animals with antrectomy and carcinogen (with or without vagotomy) developed SPEM at the base of the gastric mucosa. In association with the appearance of this metaplastic lineage, a distinct second zone of PCNA-labeled nuclei developed in the deepest portion of the mucosa. Of interest, three animals demonstrating these changes developed intestinal-type gastric adenocarcinoma. Finally, we studied the immunostaining pattern of intrinsic factor, normally a chief cell marker in rat fundic mucosa. In animals with SPEM, we observed coexpression of SP and intrinsic factor in SPEM cells at the base of the mucosa. These findings support our hypothesis that SPEM develops from a second progenitor cell population, reflecting either the unmasking of a cryptic zone or transdifferentiation of chief cells.     

Expression of a candidate marker for progenitor cells, Musashi-1, in the proliferative regions of human antrum and its decreased expression in intestinal metaplasia

AIM: Reliable makers for progenitor cells in the human stomach have not been elucidated. The aim of the present study was to clarify whether Musashi-1 (Msi-1), which has recently been proposed as a stem cell marker in mouse intestine, serves as a marker for progenitor cells in human stomach. METHODS AND RESULTS: Immunohistochemistry revealed that Msi-1+ cells were detected especially in the isthmus/neck region (the putative position of stem cells) of the adult antrum, but were limited to the basal regions of fetal pyloric glands during the early stages of development. These results suggest that Msi-1 expression occurs specifically in the stem cell-containing regions. Msi-1+ cells were intermingled with proliferating cell nuclear antigen (PCNA)+ cells in the isthmus/neck region of the adult antrum, but did not coexpress PCNA or Ki 67. Msi-1 expression overlapped partly with expression of MUC 5 AC and MUC 6, indicating that Msi-1+ cells retain some features of both foveolar and pyloric gland cell differentiation phenotypes. In contrast, Msi-1 expression in gastric glands showing intestinal metaplasia (IM) became weaker than that in the glands without IM. CONCLUSION: The specific expression of Msi-1 within the proliferative regions suggests that Msi-1 is a marker of cells with progenitor characteristics before active proliferation in human antrum.     

Cystic 'hamartomatous' gastric polyps: a disorder of oxyntic glands

In 1977, Elster et al. described gastric polyps of a new type. These polyps were formed of fundic glandular cysts in otherwise normal gastric mucosa. In this paper a series of 52 cases of this type of polyp are presented. In addition to cysts, polyps contained deformed oxyntic glands and areas where the formation of secondary irregular glands occurred from cysts and from the existing glands. However, all glands and cysts in polyps were composed of normal, but intermixed cells of oxyntic type, suggesting that deranged differentiation of otherwise normal oxyntic glands is the basic mechanism in the pathogenesis of these polyps. Based on these observations, the polyps were considered most likely to be epithelial hamartomas. In agreement with the findings of Elster et al. , the present cases showed typical clinical characteristics: three-quarters of patients were females with a peak prevalence in the fifth and sixth decades. Polyps were most often multiple and occurred only in the oxyntic mucosa of the stomach.     

Using acellular porcine limbal stroma for rabbit limbal stem cell microenvironment reconstruction

To investigate the feasibility of using acellular porcine limbal stroma for limbal stem cell microenvironment reconstruction. Limbal reconstruction was performed in rabbit partial limbal defect models. Rabbits were randomly divided into four groups: acellular porcine limbal stroma, de-epithelized rabbit limbal autograft stroma, de-epithelized porcine limbal stroma and acellular porcine corneal stroma transplantation groups. In both the acellular porcine limbal stroma and de-epithelized rabbit limbal autograft stroma groups, cornea transparency and epithelium integrity were sustained and graft rejection was not observed. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1, but it returned to K3-/P63+/Ki67+(phenotype characteristic of limbal epithelium) by postoperative months 3 and 6. In the de-epithelized porcine limbal stroma group, acute and serious immune rejection occurred by postoperative days 8-10. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1. In the acellular porcine corneal stroma group, there were some new vessel invasion into the peripheral cornea and mild corneal opacity. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative months 1, 3, and 6. In conclusion, acellular porcine limbal stroma possessed very low immunogenicity, retained a good original limbal ECM microenvironment, and thus the reconstructed rabbit limbal microenvironment maintained limbal epithelial stem cell stemness and proliferation.