Human Amnion Epithelial Cells Research Articles

Reduction of extracellular matrix protein expression in human amnion epithelial cells by glucocorticoids: a potential role in preterm rupture of the fetal membranes

Reduction of extracellular matrix protein expression in human amnion epithelial cells by glucocorticoids: a potential role in preterm rupture of the fetal membranes

The human amnion is a site of MHC class Ib expression: evidence for the expression of HLA-E and HLA-G.

The expression of HLA class I Ag by term human amnion epithelial cells was investigated. In immunostaining and FACS analysis, mAb to monomorphic class I Ag reacted extensively with amnion cells, whereas polymorphic mAb reactivity was more limited and variable. Further studies were conducted on amnion cell preparations containing negligible contaminants. Northern analysis with use of locus-specific probes demonstrated that amnion expresses two class Ib genes, HLA-E and HLA-G. Radio-immunoprecipitation with use of monomorphic mAb identified two fully glycosylated cell surface class I H chains of 44 and 41 kDa; polymorphic mAbs failed to immunoprecipitate the 41-kDa product, although 44-kDa products, typical of class Ia Ag, were identified in some preparations. Class I H chains were isolated from amnion by affinity chromatography. Microsequencing revealed that the first nine residues of the N-terminus of the 41-kDa product aligned perfectly only with HLA-E. Overall, amnion at term appears to express class Ib Ag with limited class Ia Ag. HLA-G is therefore expressed in two extrafetal epithelia: amnion and trophoblast. Identification of the class Ib protein HLA-E in amnion epithelium may have implications for preterm labor that can be associated with infection of the placental membranes.

Interleukin 4 production by human amnion epithelial cells and regulation of its activity by glycosaminoglycan binding.

The pro-inflammatory molecules, tumor necrosis factor alpha (TNF alpha), interleukin 1 (IL-1), interleukin 6 (IL-6), and prostaglandin E2 (PGE2), are postulated to have a role in human pregnancy and parturition. The ability of interleukin 4 (IL-4) to suppress the production of TNF alpha, IL-1, IL-6, and PGE2 by activated monocytes prompted us to investigate a possible regulatory role for IL-4 in human gestation. Immunohistochemical techniques were used to show that human amnion epithelium stained positively for IL-4. Tissue from both the first (n = 5) and third (n = 46) trimester expressed immunoreactive IL-4, which was detected by the use of four antihuman IL-4 monoclonal antibodies. Analysis of mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) on RNA extracts of amnion epithelial cells indicated that they were the source of IL-4. One of the anti-IL-4 antibodies used stained IL-4 protein associated with the basement membrane of the amnion epithelium. The mechanism of this association was investigated. IL-4 was shown to be a heparin-binding cytokine, which would enable it to bind to components of the extracellular matrix. Thus, this study identified a previously undescribed cellular source of IL-4, implicating a role for IL-4 in human gestation. Additionally, glycosaminoglycan binding may regulate IL-4 activity in vivo.

Type IV collagenases in human amniotic fluids and amnion epithelial cells

Gelatin zymograms revealed in human second trimester amniotic fluids a constant M r 66000 proteolytically active polypeptide that in immunoblotting was recognized by antiserum against the human fibroblast gelatinase/type IV collagenase. Isolated cultured human amnion epithelial cells produced the M r 66000 proteinase and frequently a M r 92000 one. The production of the former was enhanced by transforming growth factor-β and that of the latter by tetradecanoyl phorbol acetate. Immunoprecipitation experiments indicated that the M r 66000 amnion epithelial cell proteinase was identical with that round in the amniotic fluids and that the M r 92000 one corresponded to the human macrophage/granulocyte gelatinase/type IV collagenase. Approximate quantifications of the M r 66 000 proteinase in amniotic fluids and sera from same individuals were assessed by comparison of dilution series of the samples and standard enzyme preparations in zymography. These measurements indicated that the amount of the proteinase in the amniotic fluids was about 5–10 μg/ml representing 0.1–0.2% of total proteins. In sera the corresponding figures were 30–40 μg/ml and 0.03–0.06%, respectively. Thus the relative concentration of the M r 66000 proteinase in amniotic fluids was approx. 3–4-fold that in sera, suggesting local production of the protein. The proteinase may be produced by amnion epithelial cells that under so far unknown physiologic stimulus also may start secreting the M r 92000 enzyme.

Expression of functional insulin-like growth factor-I receptors by human amnion cells

The purpose of the study was to investigate whether amnion cells contain functional insulin-like growth factor-I receptors. To test whether human amnion cells contain insulin-like growth factor-I receptors, radioligand binding studies, affinity cross-linking studies, and Northern blot analysis were conducted in primary amnion cells and in an immortal amnion cell line (WISH). To test whether the insulin-like growth factor-I receptors on amnion cells are functional, cytochalasin B-inhibitable 2-deoxyglucose uptake was measured after stimulating the cells with insulin-like growth factor-I. Radioligand binding studies demonstrated that primary amnion cells and WISH cells contained a single class of high-affinity receptors with an apparent dissociation constant of 0.18 +/- 0.04 nmol/L and a receptor concentration of 79 +/- 26.2 fmol/mg protein and dissociation constant of 0.44 +/- 0.03 nmol/L and a receptor concentration of 33.3 +/- 6.45 fmol per 106 cells, respectively. Affinity cross-linking studies revealed two major insulin-like growth factor-I binding sites, 135 and 270 kd. Both primary amnion cells and WISH cells exhibited cytochalasin B-inhibitable tritiated 2-deoxyglucose uptake in response to insulin-like growth factor-I treatment. Finally, treatment of WISH cells caused tyrosine phosphorylation of three proteins (molecular weight, 116, 95.4, and 83.5 kd) was observed by Western blotting with antiphosphotyrosine antibodies. These results provide the first evidence that human amnion epithelial cells contain functional high-affinity insulin-like growth factor-I receptors that mediate glucose transport.

Stimulation of cultured amnion cell prostaglandin endoperoxide H synthase activity by glucocorticoids and phorbol ester

The effects of a phorbol ester activator of protein kinase C and the glucocorticoids cortisol and dexamethasone on the enzyme activity of prostaglandin H synthase (cyclooxygenase) in confluent cultures of human amnion epithelial cells were tested. Amnion epithelial cells from spontaneously delivered placentas at term were isolated and grown in culture until confluent. The activity of the enzyme prostaglandin H synthase was determined in these cells with a well-characterized enzyme assay monitoring the conversion of arachidonic acid to prostaglandin E2. The Michaelis-Menten constant and maximum velocity were determined from the substrate velocity data by means of Lineweaver-Burk plots. Amnion cells lost most of their prostaglandin H synthase activity within 2 days of culturing. This activity could be restored when cells were treated with 12-O-tetradecanoyl phorbol-13-acetate, a phorbol ester activator of protein kinase C, or with the glucocorticoids cortisol and dexamethasone. Epidermal growth factor also increased the specific activity, whereas 17 beta-estradiol had no effect on the specific activity of the enzyme. There was a direct correlation between the specific activity of prostaglandin H synthase and the output of prostaglandin E2 by cells treated with these agonists. Our data support the view that increases in prostaglandin H synthase specific activity in intrauterine tissues can be caused by stimulation by specific agonists, and this in turn is responsible for enhanced prostaglandin output by these tissues.

Human amnion epithelial cells assemble tenascins and three fibronectin isoforms in the extracellular matrix.

Monoclonal antibodies (MAb) were used to show that cultured human amnion epithelial (HuA) cells produce tenascins (Tn) and isoforms of cellular fibronectin (cFn). Tn polypeptides of M(r) 280,000 and 190,000, assembled into extracellular matrix (ECM) but not secreted into the culture medium by HuA cells, were electrophoretically similar to those produced by human fibroblasts as revealed with domain-specific MAbs. The results suggested that most Fn produced by HuA cells contained the extradomain (ED) A and an oncofetal domain but only a minor fraction EDB. In immunofluorescence Tn and Fn were seen in different cytoplasmic granules upon monensin-induced intracellular accumulation. Tn appeared to be deposited in the ECM in colocalization with Fn but distinctly slower. The present results show that cultured normal human epithelial cells synthesize Tn and three isoforms of cFn and secrete them by using different cytoplasmic pathways.

Isolation and Characterization of a Human Amnion Epithelial Cell Line That Expresses the Pregnancy-Specific β1-Glycoprotein Gene*

Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely related glycoproteins of 72K, 64K, 62K, and 54K. Together with the carcinoembryonic antigen, they form new members of the immunoglobulin superfamily. To study the molecular mechanisms that regulate expression of the PSG gene, we established a human amnion cell line, HAA58OD-8C, immortalized with an origin-defective simian virus-40 (SV40) temperature-sensitive A58 mutant virus. HAA58OD-8C cells were temperature sensitive for maintenance of transformation and expressed genes encoding PSG and the alpha- and beta-subunits of hCG. At the permissive temperature (33 C; transformed phenotype), they expressed low levels of PSG, hCG alpha, and hCG beta mRNAs and synthesized low levels of a 48K PSG polypeptide. At the nonpermissive temperature (39.5 C), HAA58OD-8C cells exhibited a differentiated phenotype, expressed increased levels of PSG, hCG alpha, and hCG beta mRNAs, and produced high levels of PSG polypeptides of 72K and 48K. Sodium butyrate induced PSG mRNA expression, and in the presence of butyrate, HAA58OD-8C cells produced high amounts of PSG polypeptides of 72K, 62K, and 48K. Ribonuclease protection analysis indicated that similar PSG transcripts were expressed by HAA58OD-8C cells and human term placenta. However, these amnion cells expressed selectively a certain population of PSG transcripts. Our results show that this amnion cell line provides a suitable model for studies of PSG gene expression and regulation.

Secretion of lysosomal hydrolases by cultured human amnion epithelial cells

Primary microcultures of human amnion epithelial cells were established, starting from sterile term placentae. Over a period of 1 week in culture, the epithelial cells release into the extracellular medium substantial amounts of some lysosomal hydrolases, such as sphingomyelinase, N-acetyl-β-glucosaminidase, α-fucosidase, β-glucuronidase, α-mannosidase, and arylsulfatase. Judging from experiments conducted with the protein synthesis inhibitor, cycloheximide, the enzymes released are not newly synthesized forms, but very likely derive from lysosomes. The constitutive secretion of lysosomal enzymes, coupled with lack of immunogenicity, makes amnion epithelial cells a convenient source of enzymes for implantation in attempts of enzyme replacement therapies.

Ed-a sequence containing fibronectin in human amniotic fluid and amnion epithelial cells

1. 1. Human amniotic fluid fibronectin (aFn) was studied by using a monoclonal antibody 52DH1 (DH) that recognizes the extra domain (ED-A) sequence of cellular Fn (cFn). 2. 2. In immunoblotting the DH antibody reacted with a sharp polypeptide band at the top of the bulk of the diffuse aFn. Another monoclonal antibody 52BF12 (BF) against the cell binding site of Fn, recognized the whole aFn. 3. 3. The ED-A sequence containing cFn (EcFn) formed a constant proportion in aFns from all amniotic fluid preparations studied. 4. 4. In amniotic membranes the DH antibody revealed bright subepithelial immunofluorescence. 5. 5. Also isolated and cultured human amnion epithelial cells were strongly positive in immunofluorescence 0and secreted EcFn into the culture medium as revealed by immunoblotting. 6. 6. The results indicate that aFn is a composition of at least two different Fn subtypes of which the EcFn most probably originates from amnion epithelial cells.

Platelet glycoprotein IIb/IIIa complex in cultured cells. Localization in focal adhesion sites in spreading HEL cells

A panel of mouse monoclonal antibodies (MoAbs) was raised that react with platelet glycoproteins (GP) IIb or IIIa. On immunofluorescence, the MoAbs reacted with 30% to 40% of the human erythroleukemia (HEL) cells. When the HEL cells were induced to spread on fibronectin in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), the MoAbs reacted with the focal adhesion sites. Only some of the GPIIIa MoAbs reacted with cells other than platelets, megakaryocytes, or HEL cells, and these showed focal adhesion sites in cultured human endothelial cells, fibroblasts, and epithelial cells from normal kidney tubules. They did not react, however, with transformed fibroblasts, fibrosarcoma cells, cultured cells from hypernephromas, or cultured human amnion epithelial cells. The results suggest that the platelet-type GPIIb/IIIa complex is only expressed in cells showing an ability to define megakaryoblastic differentiation. Localization of the GPIIb/IIIa complex at the induced focal adhesion sites in HEL cells and localization of the GPIIIa-like molecules in other cells suggest their direct role in the adhesion process and in the actomyocin organization of adherent cells.

Stimulation of Human Amnion Prostaglandin E2Production by Activators of Protein Kinase-C*

We tested the possibility that the activation of protein kinase-C by the tumor-promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol stimulates the production of prostaglandin E2 (PGE2) by the amnion. Confluent primary cultures of human amnion epithelial cells were adapted to serum-free medium and treated with the agonists for up to 8 h. Cumulative PGE2 output in the medium was measured by RIA. TPA, a potent activator of protein kinase-C, stimulated basal PGE2 output from less than 50 pg/well.5 h to 3 ng/well.5 h (P less than 0.01) in a time- and dose-dependent manner. 4-Methoxy-TPA, a weak tumor promoter derivative of TPA, was ineffective when tested in the same concentration range as TPA (1 nmol/L to 1 mumol/L). Neither calcium ionophore A23187 (20 nmol/L) nor arachidonate (1 mumol/L) stimulated PGE2 output alone, but each agonist potentiated the effect of TPA as much as 5-fold (P less than 0.01). 1,2-Dioctanoyl-sn-glycerol stimulated PGE2 output 4- to 7-fold (P less than 0.05), and this effect was potentiated by Ca ionophore and arachidonate. Studies involving actinomycin-D and cycloheximide indicated that the stimulatory effect of TPA was dependent on RNA synthesis during the first 60 min and on protein synthesis during the entire length of the phorbol ester treatment period (300 min). TPA was also able to stimulate PGE2 production after irreversible inactivation of PG endoperoxide synthase activity with acetylsalicylic acid. These results suggest that activation of protein kinase-C in amnion cells increases the de novo synthesis of the PG endoperoxide synthase enzyme in a RNA synthesis-dependent manner. Elevated intracellular calcium levels contribute to the stimulation apparently by increasing the availability of endogenous arachidonate for subsequent conversion to PGE2.

Platelet Glycoprotein IIb/IIIa Complex in Cultured Cells. Localization in Focal Adhesion Sites in Spreading HEL Cells

A panel of mouse monoclonal antibodies (MoAbs) was raised that react with platelet glycoproteins (GP) IIb or IIIa. On immunofluorescence, the MoAbs reacted with 30% to 40% of the human erythroleukemia (HEL) cells. When the HEL cells were induced to spread on fibronectin in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), the MoAbs reacted with the focal adhesion sites. Only some of the GPIIIa MoAbs reacted with cells other than platelets, megakaryocytes, or HEL cells, and these showed focal adhesion sites in cultured human endothelial cells, fibroblasts, and epithelial cells from normal kidney tubules. They did not react, however, with transformed fibroblasts, fibrosarcoma cells, cultured cells from hypernephromas, or cultured human amnion epithelial cells. The results suggest that the platelet-type GPIIb/IIIa complex is only expressed in cells showing an ability to define megakaryoblastic differentiation. Localization of the GPIIb/IIIa complex at the induced focal adhesion sites in HEL cells and localization of the GPIIIa-like molecules in other cells suggest their direct role in the adhesion process and in the actomyocin organization of adherent cells.

Oxytocin activates the inositol-phospholipid-protein kinase-C system and stimulates prostaglandin production in human amnion cells.

It has been postulated that fetal hormonal signals act upon amnion to trigger labor via prostaglandin (PG) production. Human amnion epithelial cell cultures were established to test the effects of potential activators of the inositol phospholipid-protein kinase-C effector system on intracellular inositol phosphate turnover, intracellular free calcium ([ Ca2+]i), and PGE2 production. Oxytocin provoked 3-, 2.5-, and 4-fold increases in inositol triphosphate, inositol bisphosphate, and inositol monophosphate, respectively. [Ca2+]i, measured with the fluorescent dye fura-2, was stimulated by oxytocin and vasopressin (oxytocin greater than vasopressin) in a dose-dependent manner. The [Ca2+]i transient produced by oxytocin reached a peak in 15 sec, followed by a slow return to baseline over 10 min. Preincubation with phorbol 12-myristate-13 acetate (PMA) markedly blunted the oxytocin-induced transient. No [Ca2+]i transient was seen with leukotrienes, PG, serotonin, angiotensin, or alpha- or beta-adrenergic agents. PGE2 production increased 30- to 50-fold with phospholipase-C and PMA, and 10-fold with the calcium ionophore A23187. Oxytocin and vasopressin produced 10- and 3-fold PGE2 increases, respectively. Increased PGE2 production induced by PMA, oxytocin, and A23187 was first seen after 8 hr of incubation and reached maximal levels at 24 h. Minimal PGE2 stimulation occurred with agents that produced no [Ca2+]i transient. Direct activators of the inositol phospholipid-protein kinase-C system in human amnion induce large increases in PGE2 in human amnion cells. Oxytocin and vasopressin are hormonal activators of this system in these cells, as demonstrated by their effects on inositol phosphate turnover and [Ca2+]i. These hormones also increase PGE2 production and may influence labor by stimulating PGE2 production in amnion through the inositol phospholipid-protein kinase-C system.

Adhesion of human amnion epithelial cells to extracellular matrix: Evidence for multiple mechanisms

Human amnion epithelial cells attach and flatten slowly (∼65 min) onto plastic in the presence of serum but much more rapidly (20–30 min) onto subcellular matrix (SCM) deposited by the same cells. This matrix contains both fibronectin and laminin, but neither molecule on its own can reproduce its adhesive properties. Cells attach on surfaces containing fibronectin and laminin and extend filopodial and lamellipodial areas of cytoplasm without extensive flattening in the perinuclear region. Matrix deposited onto plastic by amnion epithelial cells has trypsin-sensitive and trypsin-resistant, papain-sensitive adhesion-promoting components. Cell spreading triggered by the latter but not the former can be inhibited by pretreating the adhering cells with heparin. Other GAGs are without effect. The results are discussed in terms of multiple interactions between epithelial cells and basal laminae.

Antibodies to cytokeratin filaments in patients with alcoholic liver disease.

Previous studies have suggested that cytoskeletal elements in an altered form in vivo (alcoholic hyalin , AH) are targets for an autoimmune reaction in alcoholic liver disease (ALD). In this study we assayed autoantibodies to cytoskeletal cytoskeletal cytokeratin filaments, intermediate filaments of epithelial cells, by indirect immunofluorescence technique (IIF) using cultured human amnion epithelial cells and PtK 2 cells as targets. Sera from 20 patients with ALD, 38 patients with urogenital cancer, 22 patients with renal diseases, and from 18 healthy controls were studied. Patients with ALD had a significantly increased prevalence of cytokeratin filament autoantibodies using both PtK 2 cells (69%) and human amnion epithelial cells (69%) as substrate, as compared to other groups. The antibodies were mainly of IgA and IgM class and could be demonstrated in all forms of ALD. Interestingly, cytoskeleton antibodies have previously been observed in diseases of viral and "autoimmune" origin.

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.

Expression and distribution of vimentin and keratin filaments in heterokaryons of human fibroblasts and amnion epithelial cells.

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.

Plasmodium falciparum malaria. An amelanotic melanoma cell line bears receptors for the knob ligand on infected erythrocytes.

Erythrocytes infected with Plasmodium falciparum trophozoites and schizonts are not seen in the peripheral circulation because they attach to venular endothelium via knoblike structures on the infected erythrocyte membrane. We have recently shown that erythrocytes containing P. falciparum trophozoites and schizonts likewise attach to cultured human venous endothelial cells via knobs. In search of a more practical target cell for large scale binding studies designed to characterize and isolate the knob ligand, we tested various normal cells and continuous cell lines for their ability to bind P. falciparum-infected erythrocytes. Of the 18 cell types tested, binding of infected erythrocytes was observed to a human amelanotic melanoma cell line and amnion epithelial cells as well as to human aortic and umbilical vein endothelial cells. 96-100% of amelanotic melanoma cells bound 17+/-4 (+/-1 SEM) infected erythrocytes per positive cell, whereas fewer endothelial cells (4-59%) and amnion epithelial cells (8-19%) were capable of binding 12+/-5 and 4+/-1 infected erythrocytes per positive cell, respectively. Further studies designed to compare the mechanism of binding to the amelanotic melanoma cell line and endothelial cells showed the following results. First, that adhesion of infected erythrocytes to these two cell types was parasite stage-specific in that only erythrocytes containing late ring forms, trophozoites, and schizonts bound. Erythrocytes containing early ring forms, which do not attach to venular endothelium in vivo, did not bind to either cell type. Second, erythrocytes infected with trophozoites and schizonts of P. vivax or a knobless strain of P. falciparum, both of which continue to circulate in vivo, did not bind to either target cell type. Third, transmission electron microscopy showed that infected erythrocytes attached to the amelanotic melanoma cells via knobs. We conclude that cultured human endothelial cells and an amelanotic melanoma cell line share common determinants on their surface and that the mechanism of binding to these two different cell types is similar. The amelanotic melanoma cell line offers a useful substitute for endothelial cells in binding studies requiring large numbers of target cells.

Growth of normal human amnion epithelial cells in serum-free medium

The serum-free growth of primary cultures of normal human epithelial-like cells from amniotic membranes was accomplished. The synthetic medium consists of a 1 : 1 basal nutrient mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 supplemented with 2.5 μg/ml insulin, 50 ng/ml epidermal growth factor (EGF), 5 μg/ml transferrin, and 0.1 ng/ml triiodothyronine (T 3). EGF is the primary mitogen and is essential for cell proliferation in this system.