Human epithelial cells cultured from urine: Growth properties and keratin staining

Summary Epithelial cells can be cultured from the urine of newborn infants, providing a simple, noninvasive biopsy method. We established such cultures by standard techniques from 44% of uncontaminated, specimens obtained from newborn infants up to 1 week of age. There was an average of three colonies per milliliter of urine. Many cultures accomplished 15 to 25 population doublings in as many as five subcultures and yielded total potential culture sizes of 10⁴ to 6×10⁸ cells. Plating efficiency was high at each passage. The cultures displayed two morphologically distinct epithelial cell types. Immunofluorescent staining of keratin fibers in most of these cells further, identified them as epithelial. This work was supported by NIH grants, CA16754 (J. S. F., J. W. L.) and EY02472, AM25140, AM21358, and a Research Career Development Award (EY00125) to T.-T. S.     

Urogenital Epithelial Cells as Simple Markers of Estrogen Response in Infants: Methods and Applications

Exposure to estrogen-mimicking chemicals during critical periods of development, such as infancy, may have adverse effects. However, these effects can be difficult to characterize in most epidemiologic studies. For example, growth of reproductive organs may be susceptible to estrogenic chemicals, but measuring it requires skilled ultrasound examination; timing of pubertal onset may be altered, but observing it requires long-term follow up. To address the need for a simple marker of response to estrogenic exposures in infants, we propose a novel application of a classic marker of estrogen response in adult women: cytological evaluation of urogenital epithelial cells. In this cross-sectional study of 34 female and 41 male infants, we demonstrate that epithelial cells can be obtained from swabs of the vaginal introitus (females) and urethral meatus (males), as well as from spun urine, and that these cells respond to differential estrogenic conditions, as indicated by the relative abundance of the superficial epithelial cell type. To model varying estrogen exposure, we sampled from infants who were either newborn (highly exposed to maternal estrogens), or 12 weeks old (12W) (negligibly exposed to estrogen). Newborns had a higher percentage of superficial cells (%S), as compared to 12W (mean ± standard error: 8.3 ± 1.8 vs. 0.9 ± 0.2) (p < 0.01), consistent with an estrogen response. This difference in %S from newborn to 12W was observed similarly for swab (-7.6 ± 1.7) and urine (-7.3 ± 2.6) specimens and for males (-9.6 ± 2.9) and females (-5.2 ± 2.1). Examination of urogenital epithelial cells can successfully demonstrate estrogen response in both sexes, using cell specimens collected from either swab or urine sampling. In future studies, this simple, non-invasive method may be applied to assess whether estrogen-mimicking chemicals produce an estrogenic response in infants.     

Production and excretion of nitrate by human newborn infants: neonates are not little adults.

Endothelium-derived relaxing factor, identified as nitric oxide or its adducts, is metabolized to nitrate and excreted in the urine. Since blood pressures are lower in newborn infants compared to adults, we hypothesized that newborn infants would have increased excretion of nitrate on the day of birth. Neonatal urine was collected before 24 h of age when exogenous intake of nitrate was low. Two different analytical methods showed that nitrate accounted for >99% of nitrogen oxides in urine of healthy neonates and adults. The absolute micromolar concentration of nitrate in urine from infants was significantly below that of adults. When nitrate content was standardized for the reduced renal function in the newborn infant (creatinine content) and body mass (kilogram weight), the concentration of nitrate in neonatal urine was significantly higher than that of adults. Nitrate concentrations in the urine of prematurely born infants were twice that of nitrate measured in urine from term infants. These findings suggested that nitric oxide is produced in larger intravascular quantities in newborn infants versus adults. Thus, we postulated that nitric oxide released from a nitrosothiol would be metabolized to nitrate more readily by neonatal erythrocytes compared to red blood cells obtained from adults. Neonatal erythrocytes, suspended at concentrations of 8, 12, or 16 g per deciliter of hemoglobin, produced 1.7- to 2.1-fold more nitrate than equivalent hemoglobin concentrations of adult erythrocytes that were each incubated with S-nitroso-N-acetylpenicillamine (100 microM) over a 2-h period. Taken together, the studies of urinary nitrate in newborn infants and the ability of neonatal erythrocytes to generate nitrate are consistent with a robust production of nitric oxide immediately after birth.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Lymphocyte reactivity in pregnant women and newborn infants.

The mitotic response to phytohaemagglutinin (PHA) was determined in lymphocytes of mothers and their newborn infants obtained at delivery and seven days later by measuring the rate of 125 I-idoxuridine uptake into DNA in lymphocytes cultured in their own plasma and after washing and resuspension in fetal bovine serum. There was no difference in the unstimulated counts of maternal lymphocytes taken at delivery, whether unwashed or washed, compared with those from nonpregnant controls. With PHA stimulation the mitotic response of the maternal lymphocytes cultured in their own plasma was reduced compared with that of the control lymphocytes but washed maternal cells showed a similar response to the controls. These findings suggest that the reduced lymphocyte mitotic response to PHA in pregnancy is due to a plasma inhibitory factor This inhibition was not evident in maternal blood taken seven days after delivery. DNA synthesis in unstimulated cultures from newborn infants at birth and seven days after birth was greater than that in adult control cultures. With PHA stimulation the mitotic response of cord-blood lymphocytes cultured in their own plasma paralleled that of control lymphocytes but washed newborn cells showed a greater response. Thus plasma suppression similar to that observed in the mother seems also to affect infants at birth. This inhibition was not demonstrable in blood taken from infants of 7 days.     

Identification of cells from fetal bladder epithelium in human amniotic fluid

Summary Cultured human amniotic fluid cells consist of five different types of cytokeratin-positive epithelial cells, E-1 to E-5, differing by their size, growth morphology, and cytokeratin pattern, according to our earlier investigations. Using anticytokeratin antibodies in indirect immunofluorescence (IIF) microscopy, we show in this study that cultured urine cells contain four of the cell types found in amniotic fluid. In addition, we used two urothelium-specific antibodies, anti-UMA and anti-Las-86, in combination with cytokeratin antibodies to distinguish urothelium-derived cells in amniotic fluid and urine cell cultures. Two of the epithelial cell types were found to express urothelial antigens and thus to originate from the transitional bladder epithelium. These cells were found in 26 of the 33 amniotic fluid cell cultures and in nine of the ten urine cell cultures.     

Factors influencing the growth of alveolar type II epithelial cells isolated from rat lungs

Primary cultures of isolated alveolar type II cells have been established. Under appropriate conditions, these epithelial cells can be subcultured at least nine times. Using standard assay procedures, effects of growth factors or inhibitors can be studied. The alveolar type II cells show a marked response to both serum and growth factors in tissue culture. Either epidermal growth factor (EGF) or human urine gives an increase in thymidine incorporation (2-fold and 10-fold, respectively). The growth factor(s) in urine appears to be different from urogastrone (human EGF). The response to urine is several-fold greater than the response to a saturating concentration of mouse EGF alone. Mouse EGF added to urine does not increase the activity of urine. The period during which the alveolar type II cells respond to the growth factor(s) in urine is limited to early passages of the cells. Alveolar type II epithelial cells produce growth inhibitors in culture. Inhibitors are produced in the growth medium in either the presence or absence of serum. The concentrated inhibitor, although very unstable, gives up to a 50% inhibition of thymidine incorporation when assayed on sparse or crowded alveolar type II cells.     

The molecular cloning and expression of two CRABP cDNAs from human skin

Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). We have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABP I cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. Northern blot analysis showed CRABP II mRNA level was only slightly reduced by addition of 10(-6) or 10(-5) M RA to cultures of neonatal foreskin-derived fibroblasts, as was the CRABP I mRNA level in cultured human gut epithelial cells. In contrast, expression of CRABP II mRNA by cultured neonatal keratinocytes was strongly downregulated by RA. We conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. Our data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.     

Immunolocalization of surfactant protein-D (SP-D) in human fetal, newborn, and adult tissues.

Immunoreactive surfactant protein-D (SP-D) was assessed in human fetal, newborn, and adult tissues. In the fetal lung, SP-D was detected on airway surfaces by 10 weeks' gestation, staining increasing in the distal airways, decreasing in the proximal conducting airways with advancing gestation. In lungs from near-term infants and adults, SP-D was detected in Type II cells, serous cells of tracheobronchial glands, and subsets of cells lining peripheral airways. Immunostaining was decreased or absent in areas of lungs of neonates after injury to Type II cells, infection, or hemorrhage and was decreased in collapsed or unseptated airways from older infants with bronchopulmonary dysplasia. SP-D was also detected in many organs at all ages. SP-D was readily detected in epithelial cells and luminal material in lacrimal glands, salivary glands, pancreas, bile ducts, renal tubules, esophageal muscle and glands, parietal cells of the stomach, crypts of Lieberkuhn, sebaceous and eccrine sweat glands, Von Ebner's glands, endocervical glands, seminal vesicles, adrenal cortex, myocardium, and anterior pituitary gland. SP-D is a widely distributed member of the "collectin" family of polypeptides secreted onto luminal surfaces by epithelial cells lining ducts of many organs, where it likely plays a role in innate host defense.     

Parvalbumin, a Neuronal Protein in Brain Cell Cultures

Dissociated brain cell cultures were derived from 14-day-old embryonic as well as from newborn mice. The cells were grown in a medium containing 10% fetal calf serum. Indirect immunofluorescence was performed using antisera directed against the Ca2+-binding protein parvalbumin (Mr 12,000). In embryonic cultures a large proportion of cells was intensely stained by antiparvalbumin . In double-labelling experiments involving the simultaneous application of antisera against parvalbumin and the neuron-specific enolase, the enolase-containing cells were also parvalbumin-positive and both antisera revealed identical intracellular staining patterns. Conversely, almost no parvalbumin- and enolase-positive cells were present in cultures derived from newborn mice. However, in these cultures many cells were immunoreactive toward the myelin basic protein, an accepted marker for oligodendrocytes. The presence of parvalbumin within the embryonic brain cell cultures was confirmed by analyses of the culture extracts (4 mM EDTA, pH 7.5) by HPLC on reverse-phase supports, two-dimensional polyacrylamide gel electrophoresis, and immunoblotting. The present study suggests that in mouse brain cell cultures, parvalbumin is localized in neurons.     

Reaggregates of cells from rat testis resemble developing gonads

Reaggregates prepared from newborn rat testis cells in Moscona-type rotation cultures were analyzed and compared with normal fetal (12-21 days) and newborn testes at the light and electron microscope level. After 25 h of culture, the aggregates resembled normal testicular tissue. The cells of the surface layer were spindle-shaped and connected by adherent junctions. The epithelial cords were composed exclusively of Sertoli cells and were surrounded by elongated cells resembling the developing myoid cells in newborn testes. The basal aspect of the cords was covered by a layer of flocculent material which, in places, was organized like an ordinary basement membrane. Individual spermatogonia with pseudopodes were observed in the interstitial tissue. Some Leydig cells were organized into small clusters like those typical in newborn testes. The present observations indicate that, histologically, the reaggregation of separated testicular cells resembles the differentiation of embryonic male gonads.     

Separate isolation of Clostridium difficile spores and vegetative cells from the feces of newborn infants.

A modified taurocholate-cefoxitin-cycloserine-fructose agar medium, pH 5.5, on which vegetative cells alone could grow, was newly devised for separate isolation of Clostridium difficile vegetative cells and spores from feces. The ratio of C. difficile-positive feces from healthy newborn infants younger than 10 days of the age was 30.8%, and 93.3% of feces from healthy infants older than 20 days were positive for C. difficile. C. difficile spores alone were detected in twenty-one samples (75%) of C. difficile-positive Twenty-eight specimens. Only 10.7% (3/28) C. difficile vegetative cells alone were detected. C. difficile spores alone were detected in one of nine healthy adults. These collective results offer potential explanations for high frequent isolations of C. difficile from newborn infants without occurrence of pseudomembranous colitis.     

Glycosylation of human milk lactoferrin exhibits dynamic changes during early lactation enhancing its role in pathogenic bacteria-host interactions

Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association.     

Frequency of chromosome anomalies in children dying in the perinatal period]

348 different tissues were sampled for cultivation from 300 infants perinatally, died: a) from 118 fetuses, died at the antenatal period, 143 samples of four types of tissues were taken (kidney type -27, skin type-10, gonad type-74, blood type -32); b) 72 samples of blood and 13 samples of gonad were taken from 75 fetuses died at the intranatal period; c) 120 samples (blood type -86, gonad type -86) were taken from 97 newborn infants, died at the early neonatal period. Positive results of the growth of cultures were found in 46% (15.4% -from antenatally dead fetuses, 71.8% -intranatal deaths of infants, 64.2% -early mortality of the newborn). Among the 22 antenatally dead infants 3 appeared to have chromosome anomalies (13.6%); 1) 47, XY, +22; 2) 69, XXX; 3) 46, XX/46, XY. Among 61 intranatally dead infants 3 were found to have karyotype anomalies (4.9%): 1) 47, XX, +18; 2) 47, XY, +21;3) 46, XX/46, XY. 5 (6.5%) of the 77 newborn, dead in the first days after parturition, had the anomalies of the following types: 1) 45, XO; 2) 47, XYY; 3) 47, XY; +13; 4) 47, XY, +21; 5) 46, XX, 13q-. The total frequency of chromosome anomalies among 160 perinatally dead infants was 6.9%.     

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.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

Postnatal gender-dependent maturation of cellular cysteine uptake

Background. In view of the functional capacity of glutathione synthesis in premature infants, and because the availability of cysteine is one the rate limiting steps in glutathione synthesis, we hypothesized that the low glutathione levels in premature infants may be due to immaturity of the active cellular uptake of cysteine.

Objective. To document in cells from newborn infants the effect of maturity and gender on cysteine uptake and consequently on glutathione levels.

Methods. Incorporation of L-[35S] cysteine was measured in leukocytes from cord blood and from tracheal aspirates (TAC) of newborn infants of varying (gestational as well as postnatal) ages and gender. Cysteine uptake was correlated with glutathione in TAC.

Results. The maturity of newborn girls positively influences cysteine uptake, which is responsible for 78% of the variation in their glutathione content. However, in newborn boys, gestational and postnatal ages did not influence the cysteine uptake.

Discussion. Cysteine uptake appears to be the limiting step explaining the reported gender-related differences in glutathione as well as the low levels of this central antioxidant found in premature infants. The immature cysteine uptake found in cells from premature infants raises questions about the bioavailability of this conditionally essential amino acid in regimens of parenteral nutrition for human neonates.     

Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca²⁺-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.     

Alignment of desmosomes in stratifying human epidermis

Summary Cultured human epithelial cells stained with antibody to desmosomal proteins by indirect immunofluorescence showed linear arrays of desmosomes en face between stratified cells. To confirm that an extensive linear pattern existed on the cell surface, subconfluent cultures were viewed using scanning electron microscopy. Aligned arrays of blunt protrusions lying parallel to each other and extending in the direction of the long axis of the cell were observed on the surface of groups of superficial cells in intact cultures. That this pattern was indeed related to desmosomal distribution was verified by transmission microscopy of thin sections cut in a plane between the upper and lower surfaces of flattened stratified cells to view desmosomes directly. A similar arrangement of desmosomes was seen in intact tissue, using epidermal sheets separated from newborn foreskin. The same pattern found in flattened cells was sometimes apparent in more rounded basal cells where the cytoplasm was beginning to extend. Since desmosomal plaques are associated with keratin filaments, the alignment of desmosomes must occur in association with cytoskeletal changes as cells become flattened toward the distal epithelial surface. The primary initiation of desmosomal alignment remains to be investigated. However, the present findings demonstrate an increasingly regular membrane-cytoskeletal spatial interaction as stratified epithelial cells of skin mature.     

Laminin is produced by early rat astrocytes in primary culture

The production of laminin by early rat astrocytes in primary culture was investigated by double immunofluorescence staining for laminin and the glial fibrillary acidic protein (GFAP), a defined astrocyte marker. In early cultures (3 d in vitro; 3 DIV) cytoplasmic laminin was detected in all the GFAP-positive cells which formed the major population (80%) of the nonneuronal cells present in cultures from 20- 21-d embryonic, newborn, or 5-d-old rat brains. Monensin treatment (10 microM, 4 h) resulted in accumulation of laminin in the Golgi region, located using labeled wheat germ agglutinin. Laminin started gradually to disappear from the cells with the time in culture, was absent in star-shaped, apparently mature astrocytes, but remained as pericellular matrix deposits. The disappearance of cellular laminin was dependent on the age of the animal and the time in culture so that it started earlier in cultures from 5-d-old rat brains (5 DIV) and approximately following the in vivo age difference in cultures from newborn (12 DIV) and embryonic (14 DIV) rat brains. Our results indicate that laminin is a protein of early astrocytes and also deposited by them in primary culture, thus suggesting a role for this glycoprotein in the development of the central nervous system.