Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Isolation of functional human trophoblast cells and their partial characterization in primary cell culture

Summary Human trophoblast isolation and cell culture procedures were examined to identify variables that enhance secretion of chorionic gonadotropin (HCG) in primary culture. Brief exposure of unminced first-trimester placental specimens to a solution of trypsin-EDTA-DNAse, and isolation of the dispersed cells after Ficoll-hypaque centrifugation yielded primary cultures that were high in HCG secretion and content of epithelial-like cells. The gradual decline in HCG level with time in monolayer culture in these presumptive trophoblast cells was retarded by treatment with theophylline and cyclic adenosine monophosphate. Exposure to methotrexate (MTX) did not increase HCG secretion in normal trophoblast cells, in contrast to a 5-fold stimulation by MTX in the JAR line of choriocarcinoma cells. Clusters of polygonal cells in primary culture progressively lost their capacity to secrete HCG and their epithelial-like morphology. However, they could be maintained as cell strains through approximately 15 passages over a period of 13 to 16 weeks.     

Demonstration and maintenance of mucus secretion in cultured human gallbladder epithelial cells

Summary The method of human gallbladder epithelial cell culture has been developed successfully with active mucus secretory function. Human gallbladder epithelial cells were dissociated by Dispase digestion from the specimens obtained by cholecystectomy for uncomplicated gallbladder stone cases. The dissociated cells formed a monolayer in Eagle’fs minimum essential medium supplemented with 10% fetal bovine serum within 24 h after the inoculation. These cells were maintained for at least 2 wk without fibroblastic overgrowth. Cultured cells contained periodic acid Schiff-positive material in cellular cytoplasm for 3 d. On transmission electron microscopy these materials were identified as mucous secretory granules. Mucous secretory function was determined by [³H]glucosamine incorporation. Sixty percent of the secreted glycoproteins labeled with [³H]glucosamine was eluted in excluded fractions of Sepharose 4B gel filtration, which were considered to be mucous glycoprotein, because they were found to be resistant to proteoglycan-specific enzymes such as hyaluronidase, chondroitinase ABC, heparitinase, and heparinase. The mucous glycoprotein secretion was maintained for 3 d and found to be inhibited in a dose-dependent manner by monensin (10⁻⁷ to 10⁻⁵ M) which is a known blocker of secretory function.     

Dynamics of glial fibrillary acidic protein distribution in cultured glomerular podocytes and mesangial cells of the rat kidney

Glial fibrillary acidic protein (GFAP) has recently been shown to be expressed in the glomerular podocytes and mesangial cells (MC) of kidney (Buniatian et al (1998) Biol Cell 90, 53-61). The different localization of GFAP in podocytes and MC has raised the question whether this might reflect specific cellular functions. To address this question, in the present study podocytes and MC in early (2, 3 day-old), prolonged (5, 7 day-old) and late (14, 21 day-old) primary cultures from out-growths of glomerular explants were used. Double-immunolabeling studies demonstrated that podocytes transiently acquire myofibroblastic features, characterized by the expression of smooth muscle alpha-actin (SMAA) and increased perinuclear reaction of GFAP in prolonged cultures. The morphological differentiation of cobblestone-like podocytes into process-bearing cells was followed by loss of the myofibroblastic marker, SMAA, de novo expression of desmin, and distribution of GFAP, vimentin and desmin into the processes. In late culture, GFAP and SMAA were nearly absent from the podocytes which maintained the cobblestone-like morphology. By contrast, the myofibroblastic features gained by MC during prolonged culturing increased with time. A myofibroblast-like cytoskeleton of podocytes and MC similar to that of healthy astrocytes suggest an increased spectrum of functional activities of these cells during the acquisition of myofibroblastic features. In addition, the present study provides a new combination of biochemical and biological features by which podocytes and MC can be distinguished in culture.     

Close similarity between cultured human omental mesothelial cells and endothelial cells in cytochemical markers and plasminogen activator production

Summary The mesothelial cells obtained from human omental adipose tissue showed a typical cobblestone monolayer and reacted strongly with keratin, but did not have Von Willebrand factor. Ultrastructurally these cells revealed the existence of desmosome-like cell junctions as well as intracellular canaliculi, tubular structures surrounded by microvilli, and tonofilament-like filaments. The mesothelial cells grew much faster in the medium containing epidermal growth factor, actively took up acetylated-low density lipoprotein into their cytoplasm, and released angiotensin-converting enzyme. They also released urokinase-type plasminogen activator, but only half as much as do human umbilical vein endothelial cells; release of tissue-type plasminogen activator was not observed. Inasmuch as the mesothelial cells also released plasminogen activator inhibitor-1, as do human umbilical vein endothelial cells, we could not detect u-PA activity in culture medium. u-PA may play a role in the protection against adhesion among visceral organs. These observations indicate that cultured human mesothelial cells have characteristics closely related to those found in human endothelial cells.     

Synthesis of first trimester placental alkaline phosphatase in cultured human term placental cells

Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta.     

Ontological differences in first compared to third trimester human fetal placental chorionic stem cells

Human mesenchymal stromal/stem cells (MSC) isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC). We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous.     

Growth characteristics of human first trimester decidual cells cultured in serum-free medium: production of prolactin, prostaglandins and fibronectin

A procedure for the establishment of pure human first trimester decidual cells in primary cultures has been developed. The high rate of success in obtaining such cultures resulted from the elimination of fibroblasts by appropriate enzyme dissociation and filtration of the initial tissue sample, and subsequent maintenance of the cells in a serum-free medium supplemented with insulin, fibroblast growth factor (FGF), estradiol, progesterone, hydrocortisone, transferrin and sodium selenite. Under these culture conditions, we obtained pure and actively dividing decidual cells forming tightly packed and nonoverlapping epithelioid cell monolayers covering more than 75% of the culture area. The cultured decidual cells retained their in vivo capacity to produce prolactin and various prostaglandins (PGs), primarily PGE2. There was a marked reduction in hormone production after 20 days in culture. A massive network of fibrillar surface fibronectin was detected by indirect immunofluorescence staining of the cultured cells. The production of prolactin and PGs together with the secretion of fibronectin may play a role in the implantation and subsequent growth of the embryo. The described procedure of obtaining fibroblast-free decidual cell monolayers will promote studies on the hormonal regulation of this tissue at the time of early intrauterine life.     

A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment

Summary A method is described for the isolation of large numbers of viable disaggregated cells from human tissues. This method combined the mechanical action of a Stomacher Model 80 Lab Blender, 0.1 mg/ml trypsin or 0.5 mg/ml collagenase, and 0.1 mM [ethylene bis(oxyethylenenitrolo)]-tetraacetic acid (EGTA). Tissue (0.2 to 1.0 g) obtained from human fetal intestine, kidney, liver, lung, and skin were separately minced into approximately 1-mm³ pieces. The pieces were placed in a sterile bag containing 60 ml of calcium- magnesium-free phosphate buffered saline, the appropriate enzyme (0.1 mg/ml trypsin or 0.5 mg/ml collagenase) plus 0.1 mM EGTA, and 0.1% methylcellulose. The bag was then placed into the blender and mixed at a low speed for 3 to 20 min at room temperature. After a single cell suspension was observed by phase contrast microscopy, 10 ml of bovine calf serum was added to the cell suspension to inactivate the proteolytic enzymes. At this time 130 ml of cold Hanks' balanced salts solution containing 5% bovine calf serum was added and the entire cell suspension passed through a tissue sieve (100 mesh, 140 μm) and the cells collected by centrifugation. These cells were then resuspended into the appropriate culture medium. In comparison to other methods for establishment of cell cultures from human tissues, the method described requires shorter incubation times with relatively low concentrations of proteolytic enzymes, and yields two- to three-fold greater number of cells per tissue with 86 to 93% viability. Also, depending on the cell type, 50 to 75% of the isolated cells attached to the culture vessel within 24 h. Variation of the time and concentration of digestive enzymes can be used to select different cell types for culture. This work was supported by research grants from the National Institute of Environmental Health Sciences, Bethesda, MD (ES3101) and the United States Environmental Protection Agency, Washington, D. C. (R810146).     

Variation in the electrical properties of cultured human proximal tubule cells

Summary Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD), short-circuit current (Isc), and transepithelial specific resistance (R_T). These electrical parameters were used to determine the degree of heterogeneity among independent isolates of human proximal tubule cell cultures. Seventeen independent isolates of cells were assessed, totaling 260 individual determinations of spontaneous electrical properties. On average, these cell monolayers displayed an apicalnegative PD of 1.5 ± 0.1 mV, an Isc of 2.7 ± 0.2 μA/cm², and an R_T of 480 ± 19 ohms × cm². Each independent cell isolate, however, displayed electrical values within a narrow range, in some cases allowing isolates to be distinguished from one another. The individual isolates were also assessed for Na-coupled glucose transport, Na⁺,K⁺-ATPase activity, cAMP stimulation by parathyroid hormone (PTH), forskolin stimulation of Isc, and ouabain inhibition. With the exception of a strong correlation between Na⁺,K⁺-ATPase activity and Isc, these parameters, in contrast to electrical properties, were found to be consistent and did not reveal distinctions among the isolates. HPT cell cultures seem to consistently retain important features of proximal tubule differentiation while maintaining the variability, as demonstrated by electrical properties, that might be expected of cells isolated from a random population.     

Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

Transferrin in cultured human term cytotrophoblast cells: Synthesis and heterogeneity

Transferrin (Tf) mRNA was recently demonstrated in rat and mouseplacental tissue. Rat placental cells were shown to secrete transferrin. Thecell type with which Tf mRNA was associated was not investigated. Wetherefore studied the ability of immunopurified human term cytotrophoblastcells in culture to synthesize Tf, by means of pulse-label experiments with35S-methionine and report that these cells do synthesize Tf. Tf mRNA wasdemonstrated in the cell lysates by means of RT-PCR. Tf isolated fromcytotrophoblast and syncytiotrophoblast cells was shown to be different fromboth maternal and fetal serum Tf with respect to the distribution ofisoforms as demonstrated by means of iso-electric focusing. The iso-electricpoints were found at lower pH values (pH 5.0-5.4), compared to theiso-electric points of maternal and fetal serum Tf, suggesting a higherdegree of sialylation and glycan chain complexity.     

Irregular patterns of actin and myosin filaments in human skeletal muscle cells

Summary The ultrastructural study of cross sections of normal skeletal muscle cells showed the existence of irregular patterns of actin filaments in connection with the hexagonal pattern of the myosin filaments. The actin filaments surrounding each myosin filament vary in number from 6 to 11. The most frequent relationship is 9 to 1, followed by 10 to 1 and 8 to 1. The hexagonal pattern of actin filaments was observed only in the 6 to 1 arrays; as the actin filaments increase in number, they tend to form different polygons or circles around the myosin filaments. All described patterns may occur in each sarcomere. The actin to myosin filament ratio varies from 3 to 4 within each individual myofibril. The described variability of the actin filaments arrays leads to several difficulties in an explanation of the mechanism of muscular contraction.Director, Chief of Section, Histology. Profesor Agregado de Embriología e HistologíaProfesor Adjunto de Embriología e HistologíaResidente de Anatomía Patol'ogica de la Ciudad Sanitaria La Paz     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

Attachment and multiplication,morphology and protein production of human fetal primary liver cells cultured in hormonally defined media

Summary We established for human fetal liver cells (cultured for 2 wk) in a hormonally defined medium, optimal conditions for attachment, multiplication, and preservation of epithelial morphology as well as production and secretion of serum proteins characteristic of fetal (α1-fetoprotein, AFP) and adult (albumin and hemopexin) life. Conditions were considered optimal when cell number, albumin, and hemopexin levels were maintained throughout the 2-wk culture period. However, the decrease in AFP concentration, which occurred after a few days of culture, could not be reversed. The culture system developed is a suitable model for studying regulatory mechanisms governing structure and function during differentiation and may prove useful for testing the effect of toxic agents during fetal development of the human liver. This work was supported by the Medical Research Council of Canada (Grant MA-7768), a grant from the “Fonds de la recherche en sante du Quebec” (F.R.S.Q.) and by “CAFIR” funds from the University of Montreal. David W. Barnes is supported by NIH-NCI-grants 40475 and 01226. MS-P was recipient of a scholarship from the Notre-Dame Hospital Foundation and from the Universite de Montreal.J-FT received a summer studentship from the FRSQ and from the Canadian Liver Foundation. Presented in preliminary form at the Tissue Culture Meeting in Houston, June 1984.     

Short-Term Effects of Thyroid Hormones on Cytoskeletal Proteins Are Mediated by GABAergic Mechanisms in Slices of Cerebral Cortex from Young Rats

Thyroid hormones play important roles in brain function. However, few information is available about the effect of 3,5,3′-triiodo-l-thyronine (T₃) or thyroxine (T₄) on the in vitro phosphorylation of intermediate filament (IF) proteins from cerebral cortex of rats. In this study we investigated the involvement of GABAergic mechanisms mediating the effects of T₃ and T₄ on the in vitro incorporation of ³²P into IF proteins from cerebral cortex of 10-day-old male rats. Tissue slices were incubated with or without T₃, T₄, γ-aminobutiric acid (GABA), kinase inhibitors or specific GABA antagonists and ³²P-orthophosphate for 30 min. The IF-enriched cytoskeletal fraction was extracted in a high salt Triton-containing buffer and the in vitro ³²P incorporation into IF proteins was measured. We first observed that 1 μM T₃ and 0.1 μM T₄ significantly increased the in vitro incorporation of ³²P into the IF proteins studied through the PKA and PKCaMII activities. A similar effect on IF phosphorylation was achieved by incubating cortical slices with GABA. Furthermore, by using specific GABA antagonists, we verified that T₃ induced a stimulatory effect on IF phosphorylation through noncompetitive mechanisms involving GABA_A, beyond GABA_B receptors. In contrast, T₄ effects were mediated mainly by GABA_B mechanisms. In conclusion, our results demonstrate a rapid nongenomic action of T₃ and T₄ on the phosphorylating system associated to the IF proteins in slices of cerebral cortex of 10 day-old male rats and point to GABAergic mechanisms mediating such effects.     

Altered cytokeratin expression and differentiation induction during neoplastic transformation of cultured rat liver cells by nickel subsulfide

Rat liver T51 B cells were maintained in the presence of low concentrations of Ni(H) derived from Ni₃S₂ for 3–I5 months in culture in order to monitor cytokeratin, differentiation, and transformation patterns. Nickel exposures caused irreversible, heritable juxtanuclear aggregates of cytokeratin CKSS, which increased in size and complexity with prolonged nickel exposure, eventually resembling Mallory bodies and expressing glutamyltransferase. Altered cytokeratin expression was accompanied by induction of differentiation, with markers of both bile ductular cells and hepatocytes, such as induction of cytokeratin polypeptides CK39 and CK49, cell morphology, and cytokeratin filament network changes, whereas control cultures similarly maintained for long periods in culture remained unchanged. Altered cytokeratin expression was also accompanied by acquisition of transformation markers—loss of density dependence, progression toward calcium independence, and (benign) growth in nude mice. Observed cytokeratin aberrations may be a factor in nickel carcinogenesis, in view of the known affinity of the metal for cellular structural proteins, especially keratin, which play a role in maintenance of cell behavior.Abbreviations CK cytokeratin - GGT gamma-glutamyltransferase - HCM standard calcium medium - LCM low calcium medium (Ca²⁺ = 0.02 mM) - MB mallory body - NI neoplastic index (ratio of growth of cells in LCM/HCM) - Ni ni(II) used as leachate from Ni₃S₂ - PBS phosphate-buffered saline - SB sodium butyrate     

ADAM12 and PAPP-A: Candidate regulators of trophoblast invasion and first trimester markers of healthy trophoblasts

ABSTRACT

Proper placental development and function is crucial for a healthy pregnancy, and there has been substantial research to identify markers of placental dysfunction for the early detection of pregnancy complications. Low first-trimester levels of a disintegrin and metalloproteinase 12 (ADAM12) and pregnancy-associated plasma protein-A (PAPP-A) have been consistently associated with the subsequent development of preeclampsia and fetal growth restriction. These molecules are both metalloproteinases secreted by the placenta that cleave insulin-like growth factor binding proteins (IGFBPs), although ADAM12 also has numerous other substrates. Recent work has identified ADAM12, and particularly its shorter variant, ADAM12S, as a regulator of the migration and invasion of trophoblasts into the lining of the uterus, a critical step in normal placental development. While the mechanisms underlying this regulation are not yet clear, they may involve the liberation of heparin-binding EGF-like growth factor (HB-EGF) and/or IGFs from IGFBPs. In contrast, there has been relatively little functional work examining PAPP-A or the IGFBP substrates of ADAM12 and PAPP-A. Understanding the functions of these markers and the mechanisms underlying their association with disease could improve screening strategies and enable the development of new therapeutic interventions.