Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Long-term culture of human endothelial cells

Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10(3)/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.     

A simplified method for growth of human microvascular endothelial cells results in decreased senescence and continued responsiveness to cytokines and growth factors

Summary Human dermal microvascular endothelial cells are used to analyze the functions of microvascular endothelium in vitro. However, the low yield and short lifespan of these cells in culture has limited the types of analysis that could be performed. Human microvascular endothelial cells are typically grown in media containing supplements such as dibutyryl cyclic AMP, hydrocortisone, bovine brain extract, and antifungal agents, each of which increase the complexity of experimental design and interpretation of results. In the present study, endothelial cells were transferred after Ulex europeus-I selection into a simplified medium consisting of Endothelial Basal Medium 131, 10% fetal bovine serum, and 2 ng/ml basic fibroblast growth factor and analyzed over 3 mo. The human microvascular endothelial cells expressed the endothelial markers von Willebrand factor, CD31, P-selectin, and E-selectin. In addition, the cells showed increased proliferation in the presence of basic fibroblast growth factor (0.5 ng/ml) or vascular endothelial cell growth factor (10 ng/ml). Tumor necrosis factor-α-induced expression of E-selectin was similar in cells at Passages 3, 6, and 12, indicating that the cells maintained responsiveness to cytokines over several weeks. Furthermore, the endothelial cells attained a typical cobblestone morphology with increased cellular density and also formed capillarylike tubes on Matrigel. In summary, the human dermal microvascular endothelial cells display the expected endothelial characteristics when grown for several passages and, therefore, provide a valuable in vitro model for human microvascular endothelium.     

Isolation,growth requirements,cloning, prostacyclin production and life-span of human adult endothelial cells in low serum culture medium

Summary Endothelial cells from autopsy and biopsy specimens from a variety of adult human vascular tissue were harvested by collagenase treatment and gentle swabbing of the lumenal surface. Nutrient medium MCDB 107 containing a partially purified brain-derived growth factor (5 μg/ml), epidermal growth factor (10 ng/ml) and only 2% (v/v) fetal bovine serum supported clonal and long-term serial culture (17.6 to 26.1 cumulative population doublings) of endothelial cells from vena cava, thoracic aorta and tibial arteries at a 70% rate of success. Cumulative doublings of the cell population from eight cultures were inversely proportional to age of donor of the vascular tissue from which cells were isolated. Heparin had an enhancing effect on cell growth that varied with cell strain. Prostacyclin production of human adult endothelial cell cultures was stimulated by aracidonate and thrombin by 17 to 20 and 2 to 3-fold respectively. Endogenous and stimulated rates of prostacyclin production by human adult endothelial cells were 2 to 3 times that of human adult smooth muscle cells and 20 to 30 times that of human fibroblasts. The work was supported by Public Health Service Grant AGO3275 and Grant No. 1718 from the Council for Tobacco Research. Editor's statement This paper provides an opportunity for relatively rapid, easy growth and cloning of endothelial cells from various human specimens which are more difficult to deal with than those obtained from an intact artery or intact umbilical vein. Russel Ross     

Bovine brain and pituitary fibroblast growth factors: comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells

The mitogenic effects of brain and pituitary fibroblast growth factors (FGF) on vascular endothelial cells derived from either human umbilical vein or bovine aortic arch have been compared. Both brain and pituitary FGF are mitogenic for low density human umbilical endothelial (HUE) cell cultures maintained on either fibronectin- or laminin-coated dishes or on biomatrices produced by cultured cells such as bovine corneal endothelial cells or the teratocarcinoma cell line PF-HR-9. Pituitary FGF triggered the proliferation of HUE cells at concentrations as low as 0.25 ng/ml, with a half-maximal response at 0.55 ng/ml and optimal effect at 2.5 to 5 ng/ml. It was 50,000-fold more potent than commercial preparations of endothelial cell growth factor and 40 times more potent than commercial preparations of pituitary FGF. Similar results were observed when the effect of pituitary FGF was tested on low density cultures of adult bovine aortic endothelial cells. When the activity of brain and pituitary FGF on low density HUE cell cultures was compared, both mitogens were active. To confirm the presence in brain extract of both acidic and neutral, as well as of basic mitogen, for HUE cells, brain tissues were extracted at acidic (4.5), neutral (7.2), and basic (8.5) pH. The three types of extracts were equally potent in supporting the proliferation of either HUE or adult bovine aortic endothelial cells. When the various extracts were absorbed at pH 6.0 on a carboxymethyl Sephadex C-50 column, the neutral and basic extracts had an activity after adsorption similar to that of unadsorbed extracts. In contrast, extracts prepared at pH 4.5 lost 90-95% of their activity which was recovered in the adsorbed fraction containing FGF.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells

Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.     

Enhancement of DNA synthesis of human epithelial carcinoma cells by acidic fibroblast growth factor

Human epithelial cells that had grown out from a maxillary carcinoma were examined for their responsiveness to putative growth-controlling factors in a serum-free medium. Among the factors examined, bovine brain acidic fibroblast growth factor (FGF) at 1 to 10 ng/ml significantly promoted DNA synthesis of the cells in the presence of 5 U/ml heparin, whereas type beta transforming growth factor inhibited it in a dose-dependent manner. Fetal bovine serum at 0.6% inhibited DNA synthesis of the cells by approximately 15%, but no significant influence was observed at higher concentrations up to 10%. Epidermal growth factor, bovine pituitary gland FGF and basic FGF exhibited no significant effect on DNA synthesis of the cells. The present result suggests that acidic FGF, a known mitogen for endothelial cells, is also mitogenic for human epithelial cells derived from maxillary carcinoma.     

Transforming growth factor alpha stimulates prostacyclin production by cultured human vascular endothelial cells more potently than epidermal growth factor

Transforming growth factor alpha (TGF alpha) induces dose- and time-dependent stimulation of prostacyclin (PGI2) production by cultured human umbilical vein endothelial cells. The lowest stimulatory concentration of TGF alpha was 0.1 ng/ml and the maximal response, a 2.7-fold rise, was obtained with 10 ng/ml. The stimulation, which lasted at least 24 h, was blocked by cycloheximide and by indomethacin. TGF alpha induced PGI2 production at 10-100 times lower concentrations than did epidermal growth factor (EGF), although in stimulating endothelial cell growth the two factors were equipotent. This is the first demonstration that TGF alpha enhances PGI2 production by human cells. Moreover, this is the first evidence that it acts as both an agonist (growth) and a superagonist (PGI2 production) of EGF in the same cell type. I suggest that this phenomenon may be involved with the angiogenic activity of TGF alpha.     

Effects of an extract of human brain containing growth factor activity on the proliferation of human vascular endothelial cells in primary culture

Lesions of vascular human EC play an important role in the development of thrombi and atherosclerosis. The factors which control the repair of vascular lesions are not well known. In addition, they are difficult to study because vascular EC from large vessels are fastidious cells to grow in tissue culture. We have investigated some of the factors that may be important in human umbilical vein EC growth in primary culture. Because of reported species differences in EC culture, we have decided to culture human EC only in the presence of biological culture reagents of human origin. Human umbilical vein EC, at low seed density, can be grown to confluency on a human FN matrix or on human ECM providing the medium is supplemented with a high concentration (30%) of human serum. The optimal proliferation of EC (even when seeded at clonal density) is obtained if HBE is added. HBE cannot completely replace serum, but EC proliferate to a similar extent whether they are grown on FN or on ECM in the presence of 30% human serum of 10% human serum plus HBE. Thus, HBE contains a growth factor activity for human EC which stimulates cell growth and DNA replication. Further work is needed to purify HBE and to compare it to other endothelial cell growth factors isolated from bovine brain and bovine eye.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Stabilization by heparin of acidic fibroblast growth factor mitogenicity for human endothelial cells in vitro

The effects of heparin and other glycosaminoglycans (GAGs) on the mitogenicity and stability of acidic fibroblast growth factor (aFGF) were studied. The mitogenic activity of aFGF was assayed utilizing cultured adult human endothelial cells (AHECs) isolated from iliac arteries and veins as target cells. In most experiments, aFGF purified from bovine brain was employed; in some experiments recombinant bovine aFGF was used and qualitatively similar results were obtained. In the presence of heparin, bovine aFGF at doses between 0.5 and 1.0 ng/ml (30-60 pM) elicited half the maximum AHEC growth over a 4-day period depending on the cell line tested; in the absence of heparin, significant growth was not observed at aFGF concentrations less than 10-20 ng/ml. This effect of heparin was dose-dependent over the range 0.1-10 micrograms/ml (half-maximum dose, 2 micrograms/ml). The mitogenic activity of bovine aFGF for AHECs decreased by 50% after preincubation in culture medium without cells at 37 degrees C for 2 1/2 to 3 hours. In contrast, the mitogenic activity of bovine aFGF preincubated in the presence of heparin-containing culture medium without cells was dramatically stabilized (half-life 24-29 hours). These effects also were observed in serum-free medium. Several GAGs structurally related to heparin such as chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and hyaluronic acid neither potentiated nor stabilized aFGF mitogenic activity. However, heparan sulfate from bovine lung was found to be nearly as active as heparin in both these effects. These data suggest that the binding and stabilization of mitogens by extracellular and tissue-associated heparan sulfates might play important roles in the regulation of AHEC growth.     

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.     

Cultured endothelial cells derived from the human iliac arteries

Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins.     

An optimized culture medium for human vascular endothelial cells from umbilical cord veins

Various polypeptide growth factors, culture substrates, basal media, sera and further supplements were assayed for improvement of growth of human vascular endothelial cells from umbilical cord veins. The resulting optimized medium consisted of gelatinized culture substrates, a mixture (1:1) of Iscove's MDM and Ham's F12 basal media supplemented with 20% newborn calf serum, 500 ng/ml crude fibroblast growth factor, 20 ng/ml epidermal growth factor, 5 g/ml transferrin, 5 g/ml insulin and 10 g/ml heparin. The medium allowed long term cultivation of HUVEC up to 45 generations with maximal cell densities of about 10⁵ cells per cm² and a minimal doubling time of about 14 hours at low cell densities.Abbreviations HUVEC Human Endothelial Cells From Umbilical Cord Veins - FGF Fibroblast growth factor - EGF Epidermal Growth Factor - FCS Fetal Calf Serum - NCS Newborn Calf Serum - HBS HEPES-Buffered Saline - ECM Extracellular Matrix - LHM Peptide PyroGlu-His-Ser-Phe-Thr-Ile-Lys-Ile-ThrCONH₂ - IF 1:1 mixture of Iscove's MDM and F12 basal media     

Cytokine stimulated endothelin release from endothelial cells

Endothelin release from bovine endothelial cells of the aorta, pulmonary artery, and retinal microvessels was measured in response to various cytokines. Transforming growth factor beta (0.05-5 ng/ml) was found to be a potent stimulator (3-4 fold increase) of endothelin secretion in all three cell types. Tumour necrosis factor alpha (0.1-10 ng/ml) and interferon gamma (8-800 U/ml) had a small (1.5-2 fold increase) but significant effect on endothelin secretion from endothelial cells of large vessels but not the retinal microvessels. Interleukin-1 beta, Interleukin-6 and interleukin-8 at various doses did not affect endothelin secretion. These effects were observed at various time points from 6-24 hrs and indicate that of the cytokines tested, only transforming growth factor beta has a potent effect on endothelin release from endothelial cells of different organs.     

Serial propagation of human endothelial cells in vitro

Human umbilical vein (HUV) endothelial cells were grown for 15 to 21 passages at a split ratio of 1:5 (at least 27 population doublings) on a human fibronectin (HFN) matrix in Medium 199 supplemented with fetal bovine serum (FBS) and endothelial-cell growth factor (ECGF). This system also permitted the growth of HUV endothelial cells at cell densities as low as 1.25 cells/cm2. In addition to delaying the premature senescence of HUV endothelial cells, ECGF also reduced the serum requirement for low-density HUV endothelial-cell growth; 2.5% serum and ECGF yields half-maximum growth as compared to high serum controls. Significant HUV endothelial-cell growth was also observed in medium supplemented with either ovine hypophysectomized (HYPOX) serum, plasma-derived serum (PDS), or HYPOX-PDS in the presence of ECGF, suggesting that neither the pituitary nor the platelet contributes to HUV endothelial-cell growth.     

Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.     

Isolation and culture of human decidual capillary endothelial cells in serum-free medium supplemented with human uterine angiogenic factor

Human decidual capillary endothelial (HDCE) cells, obtained from decidual fragments of legally induced first-trimester terminations of pregnancies, were cultured in a serum-free medium supplemented with human uterine angiogenic factor (HUAF). The method of isolating the cells from the decidual tissue is described. Identification of the decidual endothelial cells was based on light- and electron-microscopic observations as well as on antifactor VIII immunoperoxidase-staining technique. The HDCE cell culture in the serum-free medium lasted for 25 weeks through eight subcultures. The cells frozen in glycerin yielded 80% viability after thawing. Electron-microscopic observations of the monolayer demonstrated Weibel-Palade bodies, desmosomes and tight junctions. HUAF is mitogenic to HDCE at 10-100 ng/ml. The dependency of HDCE cells grown in culture on HUAF may explain in part the mechanism involved in the dynamic vascular expansion occurring during gestation.