Rescue of embryonic epithelium reveals that the homozygous deletion of the retinoblastoma gene confers growth factor independence and immortality but does not influence epithelial differentiation or tissue morphogenesis

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.     

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (α-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.     

Establishment and characterization of a caprine mammary epithelial cell line (CMEC)

Summary We describe the establishment of a continuous, nontransformed cell line obtained from primary culture of a lactating (114 days postparturition) Anglo-Nubian (Capra hircus) goat mammary gland biopsy. These cells (CMEC), have been cultured in the presence of supraphysiologic concentrations of insulin and hydrocortisone for more than 560 population doublings (over 80 passages) without any sign of senescence while maintaining a normal/near-normal diploid chromosome modal number of 2n=60 and are responsive to contact inhibition of proliferation. Cytoskeletal analysis indicates that CMECs are epithelial, without detectable fibroblastic or myoepithelial cells. When grown at low density on plastic substratum, the cells tend to form island monolayer aggregates with the characteristics cobblestone morphology of epithelial cells. With increasing density, the cells organize into lumen-like structures with various morphology consisting of large and small vacuolized and nonvacuolized cells. Postconfluent cultures form epithelial raised dome-like structures, implying a process of contact-induced differentiation. This is corroborated by positive immunocytochemistry to lactation-specific proteins: β-casein and α-lactalbumin, which were predominantly expressed in dome-forming cells. We also observed an overall modulation of cytokeratin 18/19 expression associated with number of days post subculture and with the expression of lactation-specific proteins. Postconfluent cultures which contain lactation-specific, antibody-reactive, dome-like structures showed a decreased expression of keratin 18 and no (null) expression for keratin 19. Lastly, cells cultured within a collagen matrix show morphological differentiation with the organization of branching duct-like and acini-like structures. This study suggests that CMECs are a useful in vitro model for study of mammary gland development and differentiation, in particular, direct modulation of epithelial cells grown on plastic substratum or extracellular matrix without the influence of stromal elements or the necessity and variability associated with primary cell culture or tissue explants.     

Neuroendocrine cells of the human lung express substance-P-like immunoreactivity.

The occurrence of substance P (SP) in the neuroendocrine population of human lungs was investigated by immunohistochemical methods. All individuals studied (n = 16) had SP-like immunoreactive cells, being more numerous in lungs of fetuses and newborn infants than in adults. These cells, both solitary and forming neuroepithelial bodies, were found at all levels of the respiratory mucosa. Solitary neuroendocrine cells and neuroepithelial bodies were found in the bronchial and bronchiolar mucosa, while at the alveolar level neuroepithelial bodies were also seen. A more intense SP-like immunoreactivity was found in the basal cytoplasm of these cells. Occasionally they show cytoplasmic prolongations which interdigitate with neighboring epithelial cells. These facts suggest that SP-like immunoreactive cells may have a paracrine or local secretion function, acting over surrounding epithelial cells or structures situated in the lamina propria. The evidence of great numbers of SP-like immunoreactive neuroendocrine cells in fetuses and infants might be the expression of a functional role of SP in lung development.     

Human breast epithelial cells in serum-free collagen gel primary culture: growth, morphological, and immunocytochemical analysis

Human breast epithelial cells derived from various sources (fibroadenoma, reduction mammoplasty, and mastectomy tissues from premenopausal patients) have been cultured in collagen gel matrix using serum-free medium. Response to various additives has been analyzed for growth-promoting effect when added to a basal medium containing insulin, cholera toxin, and BSA. A consistent observation has been the effect of EGF and cortisol in growth stimulation of human breast epithelial cells, while separately, each additive elicited only a small response. Under this condition, employing EGF and cortisol combinations, these cells gave rise to organized colonies consisting of clusters of cells, usually spherical, without any duct-like extensions. Ultrastructural and immunocytochemical studies, using a panel of monoclonal and polyclonal antibodies, have shown that cell types and features that can be identified in the original breast tissue can also be delineated in the progeny populations. The topographical feature, consisting of lumina surrounded by a single inner layer of epithelial cells and an outer layer of basal/myoepithelial cells, can be re-created in the collagen gel system starting from small clumps of cells.     

Protein gene product 9.5 expression in the human fetal cochlea.

The distribution of protein gene product (PGP) 9.5 was analyzed in the human fetal cochlea using the indirect immunofluorescence method. In the 12- and 14-week-old human fetuses, the cells of the greater epithelial ridge and the lesser epithelial ridge were overall labelled with PGP 9.5, while the stria vascularis and the Reissner's membrane did not exhibit any staining. Spiral ganglion cells and cochlear nerve fibers were labelled with PGP 9.5 and PGP 9.5-positive nerve fibers made contact with the basement membrane of the Corti primordium in the 12-week-old human fetus. These results suggest that PGP 9.5 might be used as a histological marker of maturation and innervation in the human cochlea.     

Protein gene product 9.5 expression in the human fetal cochlea

Summary The distribution of protein gene product (PGP) 9.5 was analyzed in the human fetal cochlea using the indirect immunofluorescence method. In the 12- and 14-week-old human fetuses, the cells of the greater epithelial ridge and the lesser epithelial ridge were overall labelled with PGP 9.5, while the stria vascularis and the Reissner's membrane did not exhibit any staining. Spiral ganglion cells and cochlear nerve fibers were labelled with PGP 9.5 and PGP 9.5-positive nerve fibers made contact with the basement membrane of the Corti primordium in the 12-week-old human fetus. These results suggest that PGP 9.5 might be used as a histological marker of maturation and innervation in the human cochlea.     

Emergence of TPA-resistant ‘satellite’ cells during muscle histogenesis of human limb

Human satellite cells, obtained by surgical biopsies of traumatized legs of healthy individuals, were grown in culture in the presence of different concentrations of the phorbol ester tetradecanoyl-phorbol 12 acetate (TPA). Satellite cells, after an initial duplicative period, fused into large multinucleated myotubes which readily synthesized myosin and acetylcholine receptor (AChR). The presence of TPA at concentrations up to 10(-7) M did not affect the differentiation pattern, while higher concentrations were toxic. Thus human satellite cells are capable of differentiating in the presence of phorbol esters which block differentiation of embryonic myoblasts [1]. We then examined the appearance of TPA-resistant cells during human muscle histogenesis, since we had observed that differentiation of human myoblasts from a 6-week-old limb was completely and reversibly inhibited by 10(-7) M TPA. Differentiation of myoblasts from 6-, 7- and 8-week-old fetuses was completely inhibited by TPA. Myoblasts from 10-week-old limbs did not form myotubes in the presence of TPA; however, immunohistochemical staining with an antimyosin antibody revealed the presence of a few mononucleated myosin-positive cells which escaped the TPA-induced block of differentiation. At 12 weeks of development, a few oligonucleated, myosin-positive myotubes developed in cultures treated with TPA, and the level of AChR expressed (measured as [125I] alpha-bungarotoxin bound) reached 20% of controls. At 14 weeks of development, about half of the cells in culture were TPA-resistant and by 16 weeks of development no major differences could be detected between control and treated cells. We conclude from these data that a population of TPA-resistant myogenic cells emerges between the 10th and 14th week of human limb development and suggest that this population represents satellite cells.     

Effects of reduced calcium on proliferation and cell viability in tumorigenic and nontumorigenic rat tracheal epithelial cell lines

We have compared the in vitro growth and viability of tumorigenic and nontumorigenic rat tracheal epithelial cell lines over a range of calcium concentrations from 0.003 to 0.85 mM. A greater dependence on calcium for proliferation was seen in the nontumorigenic line as compared to the tumorigenic line at both the colony formation level and in mass cultures. In the latter culture condition, a marked differential effect on cell survival was also demonstrated. These differences in calcium dependence were seen in media containing fetal bovine serum or low concentrations of newborn calf serum and in a serum-free medium developed for these cells. The effect was also independent of the method used for calcium removal i.e., either by chelex treatment or the inclusion of EGTA. Therefore, loss of calcium dependence may be associated with tumorigenicity in rat tracheal epithelial cells offering a selectable marker for neoplastic cells in carcinogen-exposed preneoplastic cell populations.     

A quantitative matrigel assay for assessing repopulating capacity of prostate stem cells

Homeostasis of prostate tissue is maintained by stem cells, although such cells have not been well characterized. Here, we report establishment of such a method using matrigel. Matrigel containing a single-cell suspension from adult prostatic cells was subcutaneously grafted into the flank of nude mice. Prostatic duct-like structures derived from donor tissue were observed in the gel 2 weeks after transplantation. Luminal and basal cells observed in the gel expressed several markers characteristic of prostatic and/or epithelial cells. When a mixture with both EGFP-positive and negative prostate cells was transplanted, prostatic ducts consisted of either EGFP-positive or negative cells and chimeric patterns were rarely observed, suggesting that ducts were reconstituted from a single cell. Stem cell number and function were also evaluated by competition with control cells. Overall this method revealed that cells localized in the proximal portion in prostate ducts had higher reconstitution capacity than those in the distal portion. We conclude that prostate stem/progenitor cells exist and that our method is applicable to analysis of prostate stem cells, epithelial mesenchyme interactions, and prostate cancer stem cells.     

Phosphatase activity in the myenteric plexus

Intense and very intense reactions were obtained for acid phosphatase, calcium activated ATP-ase (pH 9.4), magnesium activated ATP-ase (pH 7.2) and glucose-6-phosphatase in the cytoplasms of the myenteric plexus nerve cells of the small intestine of Macacus rhesus and rabbit. Nucleotidase activity was moderate or slight and unspecific alkaline phosphatase activity absent. Both ATP-ases presented an intense activity in the myenteric plexus nerve cells of human fetuses 30, 33, and 34 weeks old; 5-nucleotidase activity, slight in the 30-week-old fetuses became more intense in the 33- and 34-week-old fetuses. The satellite neuroglial cells, nerve fibers and blood capillaries presented negative alkaline phosphatase reactions and intense or very intense activities of the other phosphatases.     

Comparative study on the appearance of various bioactive peptides in foregut derivates during the ontogenesis.

Bioactive peptides have an important multifunctional role in the gastrointestinal tract. In the present study we have investigated the dynamism of the appearance of PACAP (pituitary adenylate cyclase activating polypeptide), VIP (vasoactive intestinal polypeptide), gastrin, and secretin immunoreactivities in human foregut derivates during the ontogenesis using an immunohistochemical approach. None of these peptides were observed in the foregut derivates of an 8-week-old embryo. VIP immunoreactive nerve fibers appeared by the 11th week in the smooth muscle layers of the stomach. No other peptide immunoreactivities were observed of this stage. In 18- and 20-week old fetuses PACAP, secretin, and gastrin immunoreactive cells appeared in the developing glands of the stomach. In the duodenum gastrin immunoreactivity was present in the Lieberkühn's glands and secretin immunoreactive cells were seen between the surface epithelial cells. In the pancreas secretin immunoreactivity was found in the Langerhans islets; however, PACAP immunreactivity was observed in the exocrine portion. The distribution of VIP fibers did not change during the fetal life and it was similar to the adult pattern. According to our results the appearance of PACAP, secretin, and gastrin in the developing glands suggests their role in the proliferation and differentiation of the epithelial derivates.     

Ontogeny of the insulin response to glucose in fetal and adult sheep

The purpose of the present experiments was to examine in sheep whether the fetal insulin response to glucose was present by day 110 (d110) of pregnancy and whether the magnitude of the fetal insulin response changed between d110 and d145 (term). We also compared the responses observed in fetuses to those of adult nonpregnant sheep. Basal concentrations of glucose measured in plasma collected from the fetal femoral artery rose progressively between d110 and d145 of gestation, but did not attain the plasma glucose concentrations measured in adult sheep. Peak glucose concentrations in fetuses were achieved 10 min following the bolus injection of glucose (0.8 g/kg estimated fetal body weight) into the fetal femoral vein, and peak values increased with gestational age. Significantly higher peak glucose concentrations were achieved in adult sheep. The concentration of insulin rose rapidly in fetuses at d110, and a similar time course of insulin release in plasma was seen at all gestational ages. The peak plasma insulin concentrations were achieved at 20 min and were significantly greater in older (d140-145) than younger (d125-130) fetuses (p less than 0.05). Peak insulin values in fetuses were much less than in adult sheep. In adult sheep glucose and insulin concentrations remained elevated at 120 min following the injection of glucose, whereas in the fetus the concentration of insulin had returned to preinjection values by 60 min. The insulin/glucose ratio did not change in fetal lambs over the last one third of gestation and was not different from the adult sheep.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphogenesis of mouse mammary epithelial cells growing within collagen gels: ultrastructural and immunocytochemical characterization

Mammary epithelial cells from adult virgin mice have been cultured within collagen gels in totally serum-free medium containing either epidermal growth factor or the mammogenic hormones, progesterone and prolactin, or prolactin alone. The cellular organization, differentiation and cell-type composition of the colonies from the three culture conditions were assessed by transmission electron microscopy and light-microscope immunocytochemistry. The epithelial cells form branching duct-like structures and, when exposed to mammogenic hormones, assume a secretory morphology (including casein micelles) similar to that seen in the early to mid-pregnant mouse.     

Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro

Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.     

Degeneration and apoptosis of endometrial cells in the bitch

The relationships between changes in plasma progesterone concentrations, degeneration of the luminal epithelium, the occurrence of apoptosis of endometrial cells and endometrial leucocyte populations in the bitch were determined. Mature bitches (n = 15) were euthanized and necropsied when in diestrus (Days 7-75, n = 12) or in anestrus (Days 10, 32 and 53). Degeneration of the luminal epithelium was observed in bitches in late diestrus (Days 38-75, n = 5) when plasma progesterone concentrations were decreasing and in anestrus (Days 10 and 32, n = 2) when plasma progesterone concentrations were < 0.5 ng/mL. Endometrial leucocyte populations increased after degeneration of the luminal epithelium (around Day 42 of diestrus). Apoptosis was mainly observed in the basal glandular epithelial cells and endothelial cells of blood capillaries in all except anestrous bitches. Very few apoptotic cells were found in the superficial glandular epithelial cells and stromal cells. Higher apoptotic indices were detected in the basal glandular epithelium on Days 12-42 of diestrus than at other stages. Therefore, apoptosis of glandular basal epithelial cells occurred mainly in early diestrus, degeneration of cells of the luminal epithelium occurred from mid-diestrus to early anestrus, and the increase in leucocyte numbers may have been a consequence and not a cause of luminal epithelial degeneration.     

In vitro cytodifferentiation of perinatal rat islet cells within a tridimensional matrix of collagen

Summary Cell suspensions prepared by collagenase digestion of pancreases obtained from rat fetuses (21.5 d old) and newborns (2.5 d old) were mixed with a collagen solution and inoculated on a collagen base layer. At the onset of the culture, most acinar cells became necrotic, whereas other epithelial cells proliferated. Most of the cell clusters arranged themselves into simple polarozed structures composed of epithelial cells forming hollow spheres, and from these budded neoformed endocrine islets. Scarce fibroblasts were located close to these structures. Immunocytochemical localization of insulin and glucagon, as well as ultrastructural characteristics of the cell types revealed an intrainsular distribution similar to the in vivo localization. Tridimensional matrix of collagen offers, to perinatal pancreatic cells in culture, an environment close to the in vivo conditions: cells reorganize themselves in tissuelike structures and cell interactions concerned in the cytodifferentiation of pancreatic islets occur. This system allows for the study of undifferentiated epithelial cells—the presumed stem cells—differentiating and differentiated endocrine cells in the same preparation. B.A. is supported by a doctoral scholarship from the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture, Brussels. This work was supported by grants from the Fonds National de la Recherche Scientifique, Brussels, and from Petrofina S.A., Brussels.     

p53-dependent acinar cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas

The mechanisms linking acinar cell apoptosis and ductal epithelial proliferation remain unknown. To determine the relationship between these events, pancreatic duct ligation (PDL) was performed on p53(+/+) and p53(-/-) mice. In mice bearing a wild-type p53 allele, PDL resulted in upregulation of p53 protein in both acinar cells and proliferating duct-like epithelium. In contrast, upregulation of Bcl-2 occurred only in duct-like epithelium. Both p21(WAF1/CIP1) and Bax were also upregulated in duct-ligated lobes. After PDL in p53(+/+) mice, acinar cells underwent widespread apoptosis, while duct-like epithelium underwent proliferative expansion. In the absence of p53, upregulation of p53 target genes and acinar cell apoptosis did not occur. The absence of acinar cell apoptosis in p53(-/-) mice also eliminated the proliferative response to duct ligation. These data demonstrate that PDL-induced acinar cell apoptosis is a p53-dependent event and suggest a direct link between acinar cell apoptosis and proliferation of duct-like epithelium in duct-ligated pancreas.