Relationship between CD44 expression and cell proliferation in epithelium and stroma of colorectal neoplasms.

The cell surface glycoprotein CD44 is expressed primarily in the region of cell replication in the lower crypt epithelium of colorectal mucosa, and its expression is markedly increased in colorectal neoplasms, suggesting that expression is linked to proliferation. The association between CD44 expression and replication in individual cells was therefore analyzed by double-label immunohistochemistry for CD44 and the cell-cycle-dependent protein proliferating cell nuclear antigen (PCNA). Enhanced expression of CD44 in colorectal neoplasms occurred not only in epithelial cells but also in stromal cells, including lymphocytes and macrophages. On a topographical basis, the cellular localization of CD44 and PCNA were commonly different. Quantitatively, in all cell types studied (epithelial cells and stroma of colorectal mucosa, adenomas, and carcinomas) PCNA was present most frequently in cells lacking CD44. Statistical analysis by logistic regression models indicated that cells negative for CD44 had a higher probability of being positive for PCNA than did cells positive for CD44 (P < 0.001). These data suggest that the enhanced level of CD44 in colorectal neoplasms is asynchronous with cell replication and reflects mechanisms that act on nonproliferative stromal lymphocytes and other mononuclear cells as well as the epithelial cells.     

Ultrastructural comparison of the interface between epithelium and stroma in basal cell carcinoma and control human skin.

Comparative ultrastructural studies of basal cell carcinomas, seborrheic keratoses, actinic keratoses, and control nontumor skin from human patients demonstrate structural differences between invasive and noninvasive tumor cells. Compared to the other specimen groups, biopsies of basal cell carcinomas reveal a decrease in hemidesmosomes and an increase in actin-like microfilaments in cells at the margins of the tumors. Benign tumors, i.e., seborrheic keratoses and actinic keratoses, have hemidesmosome areas and microfilament contents resembling control nontumor skin. The most striking hemidesmosome areas and microfilament contents resembling control nontumor skin. The most striking increase in microfilaments is in the most infiltrative "morphea" variants of basal cell carcinoma. These findings, in the context of other published reports, suggest that increased microfilaments are related to enhanced motility of invasive carcinoma cells in vivo and that decreased hemidesmosomes may be related to loss of cell to substratum or "anchorage" dependence of growth in malignant cells.     

Hepatic progenitor cells in chronic hepatitis C: a phenomenon of older age and advanced liver disease

Hepatic progenitor cells (HPC) appear in a variety of liver diseases. Their occurrence in chronic hepatitis C (CHC) remains unclear, and triggering factors have to be elucidated. The presence of HPC in CHC was examined in relation to histological and virological parameters and patient age. Fifty liver biopsies of HCV-infected patients were examined. The presence of HPC was evaluated by immunohistochemical expression of keratin 7 (K7). Double immunostaining with K7 and cell proliferation marker Ki-67 was undertaken. Ductular reaction at the limiting plate, mean number of isolated progenitor cells (IPC) and isolated ductular structures (IDS) were quantified. The predominant distribution pattern of IPC and IDS and the presence of K7(+) hepatocytes were registered. Relationship between ductular reaction, IPC, IDS, presence of K7(+) hepatocytes, and patient age, hepatitis grade and stage, HCV RNA, and HCV genotype was examined. Prominent ductular reaction and increased numbers of IPC and IDS correlated significantly with older age and severe fibrosis/cirrhosis. The above HPC subtypes were not proliferating. Periportal/periseptal distribution pattern of IPC and IDS and presence of K7(+) hepatocytes were significantly more frequent in advanced hepatitis stages and in patients older than 40 years. Intraparenchymal distribution pattern correlated with younger age, lobular activity, and early fibrosis stage. K7(+) hepatocytes were encountered almost exclusively in the periportal pattern and in the presence of interface hepatitis and were more frequent among HCV genotype-1 patients. HPC activation in CHC is a common but diverse phenomenon closely related to patient age and hepatitis stage.     

Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice

The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45(-)/CD11b(-)/CD31(-)/MIC1-1C3(+)/CD133(+)/CD26(-), at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creER(T2)-R26R(YFP) mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.     

Pesticide induced marrow toxicity and effects on marrow cell population and on hematopoietic stroma

Long term inhalation of toxic pesticides used for the domestic and industrial purposes have been shown to cause moderate to severe hematotoxicity and increased incidence of several marrow degenerative diseases, specifically hypoplastic bone marrow failure condition in humans. The progression of pesticide induced hematotoxicity and the exact underlying mechanisms of toxicity that play major role in limiting normal hematopoiesis are not quite well explained. In this present study, we have developed an animal model of hypoplastic bone marrow failure following pesticide exposure to show the deleterious effects of toxic pesticides on mouse hematopoietic system. Here we have presented the results of studying long-term marrow explant culture, IL-2, IL-3 and IL-5 receptors expression profile, fibroblast colony forming unit (CFU-F), hematopoietic progenitor cell colony formation and caspase-3 expression by the bone marrow cells. We have also identified the expression levels of several extracellular apoptosis markers (CD95/Fas) and intracellular apoptosis inducer proteins (pASK1, pJNK, caspase-3 and cleaved caspase-3) in the bone marrow cells of pesticide exposed mice. The long-term marrow explant culture demonstrated the impairment in proliferation of the stromal cells/stromal fibroblasts in culture. Decreased IL-2, IL-3 and IL-5 receptors expression profile essentially hinted at the suppressed cytokine activity in the pesticide exposed marrow. CFU-F analysis showed the defect in functional maturation of the stromal fibroblasts. The decreased hematopoietic progenitor cell colony formation indicated the toxicity induced inhibition of cellular proliferation and functional maturation of hematopoietic stem/progenitor cells in pesticide exposed marrow. We have detected a sharp increase in the expression levels of both the extracellular Fas-antigen and intracellular apoptosis inducer proteins in the bone marrow cells of pesticide exposed mice that explained well, the apoptosis pathway involved following marrow toxicity. The decreased proliferation and functional maturation of marrow stromal cells and hematopoietic progenitors with subsequent increase in marrow cellular apoptosis following pesticide toxicity provided the base necessary for explaining the increased incidence of hypoplastic bone marrow failure in humans exposed to moderate to high concentrations of pesticides.     

Hepatic progenitor cells, stem cells, and AFP expression in models of liver injury

Adult hepatocytes and liver-cell progenitors play a role in restoring liver tissue after injury. For the study of progenitor cells in liver repair, experimental models included (a) surgical removal of liver tissue by partial hepatectomy; (b) acute injury by carbontetrachloride; (c) acute injury by d-galactosamine (GalN) and N-nitrosomorpholine (NNM); and (d) chemical hepatocarcinogenesis by feeding NNM in low and high doses. Serological and immunohistological detection of alpha-fetoprotein gene expression served to follow pathways of cellular differentiation. Stem cells were not required in models of surgical removal of parenchyma and in carbon tetrachloride intoxication of adult hepatocytes. In contrast, regeneration of liver occurred through biliary epithelial cells in injuries induced by GalN and NNM. These biliary epithelial cells, collectively called oval cells, are most probably derived from the canals of Hering. Proliferating bile duct cells reached a level of differentiation with reactivation of foetal genes and significant alpha-1-fetoprotein (AFP) synthesis signalling a certain degree of retrodifferentiation with potential stemness. Due to the same embryonic origin of bile ducts and hepatocytes, biliary epithelium and its proliferating progeny (oval cells) have a defined role in liver regeneration as a transit and amplification compartment. In their early proliferation stage, oval cells were heavily engaged in DNA synthesis ([3H]thymidine labelling). Pulse-chase experiments during experimental hepatocarcinogenesis exhibited their development into hepatocytes with high risk for transformation and leading to foci of altered hepatocytes. Hepatocellular carcinomas may arise either from proliferating/differentiating oval cells or from adult hepatocytes; both cell types have stem-like properties. AFP-positive and AFP-negative carcinomas occurred in the same liver. They may represent random clonal origin. The heterogeneity of phenotypic marker (AFP) correlated with a process of retrodifferentiation.     

Autologous adult rodent neural progenitor cell transplantation represents a feasible strategy to promote structural repair in the chronically injured spinal cord

Adult neural progenitor cells (NPCs) represent an attractive source for cell-based regenerative strategies in CNS disease. In animal models of spinal cord injury, syngenic adult NPCs, which were isolated from pooled post-mortem CNS tissue and co-transplanted together with fibroblasts, have been shown to promote substantial structural repair. The autologous transplantation of adult NPCs represents a major advantage compared with other sources of neural stem/progenitor cells. However, the feasibility of autologous NPC generation from a single biopsy in a relevant preclinical CNS disease model has yet to be demonstrated. To investigate this matter, adult Wistar rats underwent a cervical spinal cord lesion, which was followed by a minimal subventricular zone aspiration biopsy 2 days later. NPCs were isolated and propagated separately for each animal for the following 8 weeks. Thereafter, they were co-transplanted with simultaneously harvested skin fibroblasts in an autologous fashion into the cervical spinal cord lesion site. A total of 4 weeks later, graft survival, tissue replacement and axonal regeneration were assessed histologically. Animals receiving either allogenic NPCs combined with fibroblasts or autologous pure fibroblast grafts served as control groups. Within 8 weeks after the biopsy more than 3 million NPCs could be generated from a single aspiration biopsy, which displayed a differentiation pattern indistinguishable from syngenic NPC grafts. NPCs within autologous co-grafts readily survived, replaced cystic lesion defects completely and differentiated exclusively into glial phenotypes, thus paralleling previous findings with syngenic NPCs. The delayed transplantation 8 weeks after the spinal cord lesion elicited substantial axonal regeneration. These findings demonstrate that the therapeutic strategy to induce structural repair by transplanting adult autologous NPCs, after the successful propagation from a small brain biopsy into an acute CNS disease model, such as spinal cord injury, is feasible at the preclinical level.     

Isolation of an adult mouse lung mesenchymal progenitor cell population

Contained within the adult lung are differentiated mesenchymal cell types (cartilage, smooth muscle, and myofibrobasts) that provide structural support for airways and vessels. Alterations in the number and phenotype of these cells figure prominently in the pathogenesis of a variety of lung diseases. While these cells are thought to arise locally, progenitors have yet to be purified. In previous work, we developed a method for isolating progenitors from lung tissue: this technique takes advantage of the unique ability of cell populations enriched for somatic stem and progenitor activity to efflux the vital dye Hoechst 33342, a feature that permits isolation by flow cytometry-based procedures. Using this method, we determined that a rare population of mesenchymal progenitors resides within the CD45- CD31- Hoechst low fraction of the adult murine lung. Similar to other mesenchymal progenitors, these cells express Sca-1, CD106, and CD44; can be serially passaged; and can differentiate to smooth muscle, cartilage, bone, and fat. Overall, these findings demonstrate that a phenotypically distinct mesenchymal progenitor resides within the adult murine lung, and provide a scheme for their isolation and study.     

Hepatic stellate cell activation in nonalcoholic steatohepatitis and fatty liver

Factors associated with the development of fibrosis in nonalcoholic steatohepatitis (NASH) are largely unknown, although an association with increased hepatic iron has been suggested. Hepatic stellate cells are the principal collagen-producing cells in many liver diseases and when activated express alpha-smooth muscle actin (alpha-SMA). Hepatic stellate cell activation and association with fibrosis, necroinflammatory activity, steatosis, and stainable iron in 60 cases of NASH and 16 cases of steatosis were evaluated. All 76 patients were obese or had other risk factors for NASH. All biopsy specimens were stained for alpha-smooth muscle actin to evaluate the pattern of hepatic stellate cell activation and were evaluated for inflammatory activity (0 to 3), fibrosis (0 to 4), and stainable iron stores (0 to 4). The zonal location of activated stellate cells was recorded, and the degree of activation was graded as high-grade or low-grade based on the percentage of lobular alpha-SMA+ cells. Activated stellate cells were identified in the hepatic lobule in 74 of 76 biopsy specimens and graded as low-grade in 26 and high-grade in 48. Zone 3 was involved in 72 of 74 positive cases, and in 33 cases, the activated stellate cells were preferentially located in zone 3. The degree of stellate cell activation correlated with fibrosis but not with inflammatory activity, severity of steatosis, or stainable iron. In most cases, the degree of stellate cell activation paralleled the degree of hepatic fibrosis, but in 25 cases, the degree of hepatic stellate cell activation was greater than expected, raising the question of whether such patients are at risk for disease progression.     

Hepatic squamous cell carcinoma with hypercalcemia in liver cirrhosis

Primary squamous cell carcinoma of the liver is exceedingly rare and has previously been reported in association with hepatic teratoma, hepatic cyst or hepatolithiasis. This paper describes an autopsy case of squamous cell carcinoma which developed with hypercalcemia in a cirrhotic liver. This cancer was characterized histologically, immunohistologically and ultrastructurally, and was found to exhibit immunofluorescence positivity for anti-epidermal keratin monoclonal antibody, together with the presence of tonofilaments scattered sparsely in the cytoplasm of the cancer cells.     

Whole population cell kinetics and postnatal development of the mouse intestinal epithelium

Measurements of whole population cell kinetics of mouse intestinal epithelium during postnatal development are reported. Swiss albino mice aged 1, 2, 3, 4, 6, 8, 10, 12, 16, 19, 24, and 28 weeks were studied. Isolated epithelial preparations of jejunum and colon were used. Most kinetic parameters studied either increased or decreased with age to reach a steady level sometime after weaning. For example, before weaning about 30% of crypts were observed to be branching, while after weaning, the population of crypts that were branching decreased to adult levels of 5-10% in jejunum and 1-2% in colon. Thus, there was very active crypt formation before weaning, which likely continued into adult life but at a lower level. Villus formation appeared to be occurring in animals before weaning (i.e., 1-3 weeks), while it stopped with weaning, and thus the mean villus height increased to a plateau, which was constant with age after 4 weeks. In contrast, the mean villus width increased steadily with age. As the width of villi increased with age, the number of crypts associated with a villus also increased (presumably as a result of net crypt production in the adult). These measurements and many others (proportion of cells in S phase, number of cells/cm2, number of cells/villus, number of cells/crypt, etc.) are described.     

Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells

The potential of embryonal day (ED) 14 fetal liver epithelial progenitor (FLEP) cells from Fischer (F)344 rats to repopulate the normal and retrorsine-treated liver was studied throughout a 6-month period in syngeneic dipeptidyl peptidase IV (DPPIV-) mutant F344 rats. In normal liver, FLEP cells formed: 1) hepatocytic clusters ranging in size up to approximately 800 to 1000 cells; 2) bile duct structures connected to pre-existing host bile ducts; and 3) mixed clusters containing both hepatocytes and bile duct epithelial cells. Liver repopulation after 6 months was moderate (5 to 10%). In retrorsine-treated liver, transplanted cells formed large multilobular structures containing both parenchymal and bile duct cells and liver repopulation was extensive (60 to 80%). When the repopulating capacity of ED 14 FLEP cells transplanted into normal liver was compared to adult hepatocytes, three important differences were noted: 1) FLEP cells continued to proliferate at 6 months after transplantation, whereas adult hepatocytes ceased proliferation within the first month; 2) both the number and size of clusters derived from FLEP cells gradually increased throughout time but decreased throughout time with transplanted mature hepatocytes; and 3) FLEP cells differentiated into hepatocytes when engrafted into the liver parenchyma and into bile epithelial cells when engrafted in the vicinity of the host bile ducts, whereas adult hepatocytes did not form bile duct structures. Finally, after transplantation of ED 14 FLEP cells, new clusters of DPPIV+ cells appeared after 4 to 6 months, suggesting reseeding of the liver by transplanted cells. This study represents the first report with an isolated fetal liver epithelial cell fraction in which the cells exhibit properties of tissue-determined stem cells after their transplantation into normal adult liver; namely, bipotency and continued proliferation long after their transplantation.     

Whole population cell kinetics of jejunal and colonic epithelium in lactating dams

Previous studies make it likely that the response of the intestinal epithelium as a whole to lactation is different from that observed in the crypt population alone. We confirm this difference by whole population cell kinetics measurements of jejunal and colonic epithelium in mice that have been suckling pups for various lengths of time. We found that the fraction of cells in S phase in jejunal epithelium was significantly increased after only 1 week of lactation, maintained this elevated level after 2 weeks of lactation, but returned to normal during the third week of lactation. The cell number density in jejunum was also significantly higher after 2 and 3 weeks of lactation before returning to normal by 4 weeks. In the colonic epithelium no changes were found in the distribution of cells in G1, S, and G2 + M phases. However, a significant increase in cell number density was observed after 2 weeks of lactation, followed by a sharp decrease to a level significantly below that of normal mice after 3 and 4 weeks of lactation. We conclude that the observed significant increase in the fraction of S phase in jejunal epithelium of lactating mice is probably due to a smaller relative expansion of the villus population when compared with the expansion of the crypt population. Our data also indicate that a number of cell kinetic parameters in the intestinal epithelium of lactating mice are changing throughout the period of lactation. Thus the intestinal epithelium is probably not in a steady state during lactation.     

Decellularized human cornea for reconstructing the corneal epithelium and anterior stroma

In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro, while corneas treated with SDS supported the growth of only fibroblasts and not epithelial cells. Decellularized human corneas provide a scaffold that can support the growth of corneal epithelial cells and stromal fibroblasts. This approach may be useful for reconstructing the anterior cornea and limbus using autologous cells.     

Isolation and characterization of a stem cell population from adult human liver

Several studies suggested the presence of stem cells in the adult normal human liver; however, a population with stem cell properties has not yet been isolated. The purpose of the present study was to identify and characterize progenitor cells in normal adult human liver. By stringent conditions of liver cell cultures, we isolated and characterized a population of human liver stem cells (HLSCs). HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, and CD90 but not the hematopoietic stem cell markers CD34, CD45, CD117, and CD133. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin-19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed albumin, alpha-fetoprotein, and in a small percentage of cells, cytokeratin-8 and cytokeratin-18, indicating a partial commitment to hepatic cells. HLSCs differentiated in mature hepatocytes when cultured in the presence of hepatocyte growth factor and fibroblast growth factor 4, as indicated by the expression of functional cytochrome P450, albumin, and urea production. Under this condition, HLSCs downregulated alpha-fetoprotein and expressed cytokeratin-8 and cytokeratin-18. HLSCs were also able to undergo osteogenic and endothelial differentiation when cultured in the appropriated differentiation media, but they did not undergo lipogenic differentiation. Moreover, HLSCs differentiated in insulin-producing islet-like structures. In vivo, HLSCs contributed to regeneration of the liver parenchyma in severe-combined immunodeficient mice. In conclusion, we here identified a pluripotent progenitor population in adult human liver that could provide a basis for cell therapy strategies.