Human Amniotic Fluid Cells Research Articles

Fragments of Myelin Basic Protein Derived from Human Brain are not Mitogenic in Cultures of Human Amniotic Fluid Cells and Other Cells

The possible mitogenic effects of a number of preparations of the myelin basic protein (MBP) of human brain have been investigated in various cell types in culture, including human amniotic fluid cells. Intact human MBP, as well as fragments derived by BNPS-skatole and cathepsin D treatment, and isoelectric focusing fractions of human MBP, showed no mitogenic activity. These results are consistent with recent findings that the fibroblast growth factor (FGF) activity of bovine brain does not originate from MBP.

Inability of second trimester human amniotic fluid cell cultures to aromatize C19-steroids.

Amniotic fluid (AF) and fibroblast (F) cell types, derived from human amniotic fluid by amniocentesis in the second trimester, were cultured using media of varying composition and their ability to transform C19-steroids to oestrogens was investigated by fluorimetry, radioimmunoassay, tritium release and measurement of cytochrome P450. Although the cells were shown to be metabolically active by the presence of mitotic figures, the incorporation of tritiated leucine into protein and the production of the beta-subunit of hCG, no evidence of aromatization was obtained despite the inclusion in the medium of a number of steroids known to be transformed to oestrogens in several in vivo and in vitro preparations. The addition of reported stimulants of aromatase activity, viz. hCG, FSH, diethylstilbestrol, prostaglandins E2 and F2 alpha and dibutyryl cyclic AMP, was without effect.

Metabolism of [4-14C]androstenedione by cells cultured from human amniotic fluid.

The conversion of [14C]androstenedione by human fibroblast (F) and amniotic fluid (AF) cells obtained by amniocentesis was investigated using human dermal fibroblasts (DF) as controls. Cell suspensions were incubated with [14C]androstenedione in the presence of a NADPH generating system. Steroid reaction products were separated from unreacted substrate by chromatography on micro-columns of magnesium oxide, partially resolved by partition chromatography on celite and further characterized by thin-layer chromatography and recrystallization to constant specific activity. In the case of F cells the pattern of metabolism was qualitatively similar to that of DF cells; the predominant metabolite was testosterone and several uncharacterized metabolites were detectable. However testosterone was the only metabolite isolated from incubations with AF cells. The results demonstrate distinct differences in the capacity of AF and F type cells to metabolize [14C]androstenedione and support the view that F cells resemble typical fibroblasts from dermis or other connective tissues.

Flow cytometric analysis of small DNA content differences in heterogeneous cell populations: human amniotic fluid cells.

Accurate flow cytometric determination of nuclear DNA fluorescence has been attempted with human amniocentesis materials employing a detergent nuclear isolation protocol and 4,6-diamidino-2-phenylindole (DAPI) staining. Assay of native fluids yielded inadequate fluorescence histograms, whereas analysis of cultured amniocytes yielded unimodal histograms in 80 of 83 samples. Three fluids displayed unexpected bimodality, which may have resulted from the interaction between detergent and chromatin in rare cell types. Using a standardized protocol for preparation, staining, and assay, including brief periods of cocultivation of a human triploid reference standard, the percent experimental error in the flow cytometric DNA content estimate was as low as 0.2% in repeated assays of a single sample, and 0.4% in assays of a given genotype at different occasions. In two series of blindly assayed specimens, the percent standard error of the DNA content estimate ranged from 0.6 to 1.0%.

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers.

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.

Identification of cells from fetal bladder epithelium in human amniotic fluid.

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.

Characterization of hCG regulation in cultured human amniotic fluid cells: II. Mechanisms for stimulation.

We showed previously that sodium butyrate stimulated human chorionic gonadotropin (hCG) measured by radioimmunoassay of medium from human second trimester amniotic fluid cell cultures, termed AF cells. We now find that stimulation of hCG in the presence of sodium butyrate takes as long as 20 h. When AF cells are preincubated with sodium butyrate, hCG levels increase in direct relation to length of the preincubation period. These findings suggest that elevation of hCG is not due merely to a release of hormone from the cells. Addition of cycloheximide or Actinomycin D inhibited protein synthesis and RNA synthesis, respectively, and prevented the stimulation of hCG by sodium butyrate. These results lend support for a mechanism of regulation involving protein and RNA synthesis, the increase in hCG levels being due to new synthesis of the hormone. Other agents reported to influence hCG production by different types of cell cultures include dibutyryl cyclic AMP, epidermal growth factor (EGF), methotrexate, and hydroxyurea. Dibutyryl cyclic AMP and EGF have no effect on hCG production in our AF cells: methotrexate causes a minimal increase, hydroxyurea causes a further increase, but sodium butyrate has the strongest stimulatory effect. We conclude that amniotic fluid cells in culture are susceptible to environmental agents capable of modulating synthesis of hCG by mechanisms involving synthesis of RNA and protein.

Decreased oxygen supply enhances growth in culture of human mid-trimester amniotic fluid cells.

Human mid-trimester amniotic fluid cells were cultivated under conditions of decreased oxygen supply. Compared to control cultures the low-oxygen group showed improved growth which was quantitated by three independent assays (1) direct cell counts, (2) bromodeoxyuridine (BrdU)-Hoechst flow-cytometry, and (3) cloning efficiency. The growth promoting effects of lowered oxygen hold for all major morphologic categories of amniotic fluid cells.

HLA-D and -DR antigens on human amniotic fluid cells. I. Lack of expression of HLA-D.

Human amniotic fluid cells, known to express HLA-A, -B, and -C antigens, were tested for the presence of lymphocyte-stimulating antigens (LD or HLA-D) using modifications of the mixed lymphocyte culture (MLC) and primed lymphocyte typing (PLT) tests. Peripheral blood lymphocytes were co-cultured with various concentrations of allogeneic amniotic fluid cells, either growing as a monolayer culture in microtiter plates or suspended in medium following treatment with trypsin. The kinetics of such mixed lymphocyte amniotic fluid cell culture (MLAC) reactions were followed during days 3 to 8. Under none of these conditions did amniotic fluid cells significantly stimulate allogeneic lymphocytes, even after lymphocytes were specifically primed in the PLT assay to the HLA-D antigens segregating in the family of the amniotic fluid cell donor. Furthermore, in three-cell experiments, amniotic fluid cells failed to inhibit an ongoing MLC reaction, indicating that the absence of proliferative response to amniotic fluid cells is not due to active suppression. Taken together, these data strongly suggest that amniotic fluid cells either do not express HLA-D antigens or do not express them in a form that is detectable in either primary or secondary MLC.

Fluorimetric assay of phosphate-activated glutaminase

A fluorimetric assay for the estimation of phosphate-activated glutaminase is presented. The liberated glutamate is separated from glutamine using a Dowex centrifugation technique allowing multiple samples to be rapidly analyzed. Glutamate is estimated fluorimetrically by reaction with o-phthaldialdehyde. Parameters for the assay were worked out based upon characterization of human frontal cortex glutaminase. High phosphate-activated glutaminase was found in cultured human skin fibroblasts and amniotic fluid cells and rat frontal cortex and striatum. Human caudate nucleus and frontal cortex activity was variable, but related in an exponential manner. Human and rat liver activity was markedly lower than brain activity.

HLA typing of cultured amniotic fluid cells.

HLA typing was performed on 18 cultures of human amniotic fluid cells using cytotoxicity and absorption technics. Confirmation of antigen assignments was obtained in nine of ten instances, where HLA typing also was performed on cord blood. Three major problems were encountered in performing these studies: (1) complement cytotoxicity, (2) false-positive reactions, and (3) false-negative reactions. False-positive and false-negative reactions occurred more frequently with sera defining HLA-B locus specificities than with sera defining HLA-A locus specificities. Absorption studies were helpful in making antigen assignments when false reactions occurred. Preliminary studies suggest that the frequency of false-positive reactions can be decreased by absorbing HLA typing sera with antigen-negative amniotic fluid cultured cells, buffy coat, or platelets. Accurate antigen assignment is difficult when parental HLA types are unavailable.

Succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells. A methodological study.

Through a methodological evaluation, reliable histochemical and biochemical methods for succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells were developed. The histochemical method includes a cleaning of the cultured cells in 1 mM malonate in 0.9% NaCl, air-drying and fixation in acetone (5 min at -20 degrees C), coating of cells with CoQ10 (0.2 mg/ml in ether/acetone) and incubation for 1 h at 37 degrees C in 50 mM succinate and 0.5 mg/ml Nitro BT in 200 mM phosphate buffer, pH 7.6 PMS as an intermediate electron carrier was found inferior to exogenous CoQ10. Both types of cells exhibit equal activity. In the biochemical method homogenizing was performed in 50 mM Tris-HCl buffer, pH 7.5, and 200 mM sucrose. The standard incubation was 2.0 mM INT and 10 mM succinate in 10 mM Tris-HCl buffer, pH 7.5 for 1 h at 37 degrees C. The apparent Km values for INT and succinate were estimated to 0.39 mM and 0.13 mM, respectively, while I0.5 for malonate was 0.46 mM. Activity in amniotic fluid cells was 18.1 pkat/mg protein and in human skin fibroblasts 20.3 pkat/mg protein. Specificity of the methods was tested by use of a Chinese hamster fibroblast strain B9 known to be succinate dehydrogenase deficient in addition to various control experiments. Congruent results were obtained with the two methods.

Metaphase chromosomes from primary culture of human amniotic fluid cells

A new growth medium is specially designed for reliable and reproducible primary culture of human amniotic fluid cells. In comparison with many conventional media at 15 to 20% serum, this medium at 4% serum provides actively growing primary cultures for cytogenetic studies in less time with a larger number of metaphase cells present.

Determination of pyruvate dehydrogenase in cultured human fibroblasts and amniotic fluid cells

A new assay method for pyruvate dehydrogenase in platelets [1] using in situ generation of [1- 14C]pyruvate from [1- 14C]lactate has been further developed for the use in cultured human fibroblasts and amniotic fluid cells. A very low blank rate was found compared to the method using [1- 14C]pyruvate as substrate and an activity of 20 times the blank was obtained in normal fibroblasts and amniotic fluid cells. The pyruvate dehydrogenase was linear with time and protein concentration. The activity was greatly decreased by addition of non-radioactive pyruvate to the assay mixture and by preincubation with ATP. The cofactor requirement was similar to pyruvate dehydrogenase from other sources. Normal activities in cultured human fibroblasts and amniotic fluid cells were 13.9 pkat/mg protein ( n = 15) and 21.7 pkat/mg protein ( n = 10), respectively. The method was found to be very reliable and makes the diagnosis of pyruvate dehydrogenase deficiency possible in easily accessible tissue such as cultured fibroblasts. Prenatal diagnosis may also be a possibility.

Human amniotic fluid cells grown in a hormone-supplemented medium: suitability for prenatal diagnosis.

A new supplemented medium has been developed to improve human amniotic fluid cell growth and to reduce the dependence on exogenously added serum. The medium consists of a mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with Hepes, antibiotics, and 10 growth-promoting factors at 4% fetal bovine serum. Good clonal growth is achieved consistently in 8--13 days and is associated with large numbers of metaphase cells. Primary clones may be analyzed directly, thereby reducing difficulty with interpretation of chromosomal mosaicism. This medium could also be used for cultivation of fetal solid tissues and peripheral blood cultures of lymphocytes.

Influence of extracellular matrix on the proliferation of human amniotic fluid cells in vitro.

Factors which influence the proliferation of human amniotic fluid cells in vitro have potential importance in reducing the time for prenatal diagnosis. Fibroblast growth factor (FGF) has been shown to be mitogenic for human amniotic fluid cells. The observation that cells which respond to FGF in vitro produce their own extracellular matrix (ECM), led to the use of an ECM as a substrate to assess proliferation. Pooled amniotic fluid cells maintained on an ECM prepared from bovine corneal endothelial cells demonstrated a significant increase in proliferation when compared with cells maintained on plastic substrate in the presence or absence of FGF. If FGF was added to cultures of amniotic fluid cells maintained on ECM, further increases in proliferation were noted compared with cells maintained on ECM in the absence of FGF. These results indicate that the substrate upon which amniotic fluid cells are maintained can have a profound influence on their proliferation.

Effects of Econazole, Fungizone and Pimafucin on cell growth, lysosomal enzyme activity and sulphate metabolism of cultured human skin fibroblasts and amniotic fluid cells.

Human skin fibroblasts and amniotic fluid cells showed sensitivity to antifungal agents in the order Econazole less than Pimafucin less than Fungizone, with the last initially reducing cell growth 10-20%, even at the level recommended for cell culture. The activities of the lysosomal enzymes alpha-L-fucosidase, beta-D-glucuronidase and alpha-D-mannosidase were unaffected, even by high concentrations of all three antifungal agents. Sulphate incorporation by cultured fibroblasts was increased by removal of sulphate salts and Crystamycin (contains streptomycin sulphate) from the culture medium. Sulphate incorporation into and degradation from macromolecules was only slightly reduced by antifungal agents. Human skin fibroblasts and amniotic fluid cells can be cultured long term in the presence of Econazole or Fungizone and used for lysosomal enzyme assay and sulphate kinetic studies.

4] Preparation and characterization of procollagens and procollagen-collagen intermediates

This chapter describes specific methodology for the purification of the five different procollagen types both from tissues and from sources that synthesize these proteins in vitro. Smooth muscle cell cultures are chosen to isolate procollagen because the cells are easily propagated in tissue culture, they produce relatively large amounts of types I and III procollagen, and they alsosynthesize small but detectable amounts of types IV and V procollagen. Types I and III procollagen purification involves: isolation of smooth muscle cells; metabolic labeling; and ion-exchange chromatography. Because fractionation on DEAE-cellulose is performed under conditions in which procollagen retains its native conformation, it is critical, after removal of the labeled culture medium from the cells, to perform all procedures at 0–4 ° to prevent denaturation of the protein. Type II pro T collagen is prepared from chick sternal cartilages or from matrix-free cartilage cells. After removal of sterna the cells were filtered through lens paper and centrifuged. The culture medium was processed and chromatographed on DEAE-cellulose. Type II procollagen is migrated on SDS-PAGE similarly to type I procollagen before reduction and to the pros α(I) chain after reduction. Type IV procollagen can be prepared and characterized predominantly from three biosynthetic systems: the EHS sarcoma, human amniotic fluid cells, and rat parietal yolk sac. Procedures for isolation of type IV procollagen are the same as those just described for types I and III. In type V(AB) rocollagen purification are excised from fifty 19-day-old chick embryos and incubated in DMEM. Labeling is performed using 50 μCi of [3H]proline. To remove any precipitated material and was chromatographed on a DEAE-cellulose column.

Stimulation of human amniotic fluid cell proliferation and colony formation by cell plating on a naturally produced extracellular matrix

AbstractHuman amniotic fluid cells exhibit a higher cloning efficiency and rate of cell proliferation when maintained on dishes coated with a naturally produced extracellular matrix (ECM) in comparison with the regular tissue culture plastic. In 22 out of 31 amniotic fluid samples there was by plating the cells on ECM a 2–6 fold increase in number and size of colonies and in the cell density per colony as detected by actual staining and viewing of each colony. These effects yielded, in 21 of 41 additional samples, a reduction ranging from 2–8 days, in the culture time elapsing between amniocentesis and the first harvesting of cells for chromosomal analysis. An even greater effect was obtained with primary cells that failed to attach to plastic surfaces and stayed floating in the medium but did attach and proliferate when seeded on ECM. Cells that were left firmly attached to ECM after the first trypsinization and harvesting of cells for chromosomal analysis yielded colonies ready for second karyotyping in less than half the time required for cells maintained on plastic. Studies with secondary cultures of human amniotic fluid cells have demonstrated a 5–10 fold decrease in serum requirement of cells cultured on ECM as compared with plastic. Addition of fibroblast growth factor (FGF) to the cultures further potentiated the effects of ECM. The ECM induced stimulation of cell attachment and proliferation was not associated with any chromosomal anomalies, nor did it interfere with the handling procedure. ECM coated dishes may be useful to reduce the time interval between amniocentesis and diagnosis, in particular when the amniotic cells exhibit an exceedingly slow rate of proliferation on plastic or when large quantities of cells are required for enzymatic studies.

ANDROGEN METABOLISM AND SPECIFIC 5α-DIHYDROTESTOSTERONE (DHT) RECEPTORS IN HUMAN AMNIOTIC FLUID FIBROBLASTS

This study was designed to find out whether human amniotic fluid cells have steroid 5 α-reductase activity and specific intracellular androgen receptors, and whether they could be used for antenatal diagnosis of male pseudohermaphroditism due to androgen resistance. "Fibroblast-like" cells cultures were raised from 12 amniotic fluids collected at mid-gestation. Specific receptor bound-DHT in the cell lysate was measured by a charcoal adsorption method. The binding specificity of the DHT-receptor was investigated. Physico-chemical properties of the binding component were studied by Sephadex G25 column chromatography, by density gradient ultra-centrifugation and by polyacrylamide gel electrophoresis. The dissociation constant of the 3H-DHT receptor complex varied between .38 and 2.10 × 10-9M and the number of binding sites ranged from 110 to 644 fmoles.mg DNA. Moreover, amniotic fluid fibroblasts were capable of metabolizing androgens : when the cell were incubated with testosterone, DHT and Androstane-diols were measured by gas liquid chromatography. 5 α-reductase activity and high-affinity specific DHT binding protein are expressed in amniocentesis cells.From these data, it is thus possible to identify, prior to birth, individuals with 5 α-reductase deficiency or with no detectable androgen receptor.