Ductular morphogenesis and functional polarization of normal human biliary epithelial cells in three-dimensional culture

BACKGROUND/AIMS: The understanding of the physiology and function of human biliary epithelial cells (hBEC) has been improved by studies in monolayer culture systems. The aim was to develop a polarized model to elucidate the mechanisms of ductular morphogenesis and functional differentiation of hBEC. METHODS: The morphological, phenotypic and functional properties of hBEC cultured as three-dimensional aggregates in collagen gel were assessed in medium supplemented with (or without) human hepatocyte growth factor (hHGF) and foetal bovine serum. RESULTS: In the absence of added mitogens and serum, cells maintained as morphologically polarized aggregates, organized around a central lumen, were positive for phenotypic markers of biliary epithelium and negative for markers of other cell types. Functional markers, gamma-glutamyl-transferase, anion exchanger-2, responses to gamma interferon and forskolin induced secretion, were preserved. hHGF increased both the size and number of aggregates and induced hBEC to invade the gel and lumena forming anastomosing networks of cells. CONCLUSIONS: Collagen gel culture in the absence of added growth factors and serum provides a model for analysis of the polarized functions of hBEC. The formation of poorly organized cords of cells in response to hHGF suggests that collagen gel culture may provide a model for the investigation of atypical ductular morphogenesis of the human biliary tract.     

Proliferation and differentiation of canine prostatic epithelial cells in culture

When cultured in monolayers, non-secretory epithelial cells from canine prostates actively synthesize DNA, RNA and proteins; subsequently, mitotic figures and an increase in cell number are observed. During this culture period, cell size and acid phosphatase activity remain constant. As the culture proceeds, these cells mature into secretory cells as evidenced by a gradual shift in their density in Percoll gradients to the density of secretory cells and by an increase in the cellular content of acid phosphatase. During maturation, the size of the non-secretory cells increases and their morphology changes and becomes similar to that of the secretory cells. When a homogeneous population of secretory cells is cultured, DNA synthesis is minimal and few mitotic figures may be observed while cell number and cell density remain constant. Early in the culture period, their size increases and by 2 weeks their acid phosphatase activity is 2--3-fold higher than that of the non-secretory cells. Thus, upon culture, the non-secretory epithelial cells enter and proceed through the cell cycle with evidence of DNA synthesis and mitosis. Those cells leaving the cycle undergo maturation into secretory cells which further differentiate with the concomitant appearance of acid phosphatase activity. This model will be useful to study prostatic hyperplasia and hypertrophy and the control mechanisms involved in these phenomena.     

人参皂苷预处理对电离辐射下人造血干细胞的保护作用及其机制探讨

目的观察人参总皂苷预处理对电离辐射下人造血干细胞(HPCs)的保护作用,并探讨其作用机制。方法将HPCs随机分为对照组和观察组,对照组不处理,观察组予以人参皂苷5.0μmol/L培养24 h,分别用剂量为0、1、2、5 Gy的X线对两组细胞进行照射。MTT法测算细胞存活率,流式细胞仪测定辐射后24 h细胞内的活性氧(ROS),Western blot法测定细胞内的Caspase-3和Nrf-2。结果随着辐射剂量的增加,两组细胞存活率均逐渐下降(P均<0.05),细胞内ROS活性增强、Caspase-3表达增加、Nrf-2表达减少(P均<0.05);与相同照射剂量对照组比较,观察组细胞存活率增加(P均<0.05),细胞内ROS活性减弱、Caspase-3表达减少、Nrf-2表达增加(P均<0.05)。结论人参皂苷对电离辐射下的HPCs具有保护作用,该作用与其抗凋亡和抑制氧化应激有关。     

Primary culture of human gallbladder epithelial cells

Growth requirements of epithelial cells isolated from the human gallbladder were examined. The growth of gallbladder epithelial cells (GBEC) was stimulated by medium conditioned with gallbladder fibroblasts (CM-GBF) in a dose-dependent manner. The conditioned medium derived from human embryo lung fibroblasts also showed similar growth stimulating activity for GBEC, suggesting that fibroblasts secrete a factor or factors which induce GBEC growthin vitro. CM-GBF in the presence of 10% fetal bovine serum increased GBEC growth up to 10 times the growth in the absence of CM-GBF supplement. GBEC cultured with CM-GBF showed hexagonal shape under a phase-contrast microscope, and also expressed cytokeratin and mucopolysaccharide in the cytoplasm, both of which are specific for GBEC. Electron microscopy revealed desmosomes and tight junctions between the cells and microvilli on the apical plasma membrane, suggesting that they regained morphological polarity in the medium containing CM-GBF. These results shows that CM-GBF is essential for the growth and the differentiation of GBEC in culture.     

Androgen-induced cell growth and c-myc expression in human non-transformed epithelial prostatic cells in primary culture.

We assessed androgen-induced cell growth and c-myc expression in human non-transformed epithelial prostatic (HNTEP) cells in primary culture. Prostatic tissue was obtained from 48 retropubic prostatectomy patients (age: 61-77years) with benign prostatic hyperplasia (malignant tumors excluded). HNTEP cells were treated with testosterone or DHT, alone or in association with hydroxyflutamide. DHT action on c-myc mRNA was examined using Northern blots and RT-PCR. RT-PCR also was used to verify if HNTEP cells expressed the androgen receptor gene. Cell proliferation was assessed on days 3 and 6. Testosterone (2 x 10(-11) M) and DHT (10(-13)M) caused a significant increase (P < 0.05) in cell proliferation on both days. Addition of hydroxyflutamide (10(-6) M) to DHT abolished cell proliferation. HNTEP cells expressed androgen receptor (AR) gene and the treatment with DHT increased AR mRNA levels. C-myc expression was maximal at 30 min and 1 h with DHT (10(-13) M). C-myc seems to play a key role in the control of hormone responsiveness and cell proliferation in epithelial prostatic cells. The detection of androgen receptor gene expression and the increase in this expression with the addition of androgen shows that the HTNEP cells maintain functional characteristics and hormone dependence, and that they are a fruitful in vitro model for studying steroid hormone action mechanisms.     

Primary culture of human gallbladder epithelial cells.

Growth requirements of epithelial cells isolated from the human gallbladder were examined. The growth of gallbladder epithelial cells (GBEC) was stimulated by medium conditioned with gallbladder fibroblasts (CM-GBF) in a dose-dependent manner. The conditioned medium derived from human embryo lung fibroblasts also showed similar growth stimulating activity for GBEC, suggesting that fibroblasts secrete a factor or factors which induce GBEC growth in vitro. CM-GBF in the presence of 10% fetal bovine serum increased GBEC growth up to 10 times the growth in the absence of CM-GBF supplement. GBEC cultured with CM-GBF showed hexagonal shape under a phase-contrast microscope, and also expressed cytokeratin and mucopolysaccharide in the cytoplasm, both of which are specific for GBEC. Electron microscopy revealed desmosomes and tight junctions between the cells and microvilli on the apical plasma membrane, suggesting that they regained morphological polarity in the medium containing CM-GBF. These results shows that CM-GBF is essential for the growth and the differentiation of GBEC in culture.     

A monolayer culture of human gastric epithelial cells

Our aim was to develop a fibroblast-free monolayer culture of human gastric mucosal cells, using the specimens obtained by routine endoscopic biopsy. Human gastric mucosa obtained from normal volunteers by endoscopic biopsy was dissociated from collagenase and hyaluronidase. Dissociated cells were cultured in supplemented Coon's modified Ham's F-12 medium. Within 24 hr of inoculation, the cells were attached to the culture dishes. This was followed by cellular outgrowth. On phase-contrast microscopy, all cells had epithelial characteristics and fibroblasts were not observed. Ninety percent of cells contained periodic acid Schiff reaction-positive mucous granules after diastase digestion consistent with mucous epithelial cells. Two percent of the cells gave a strong reaction for succinic dehydrogenase activity (parietal cells). Immunohistochemical staining for pepsinogen in cultured cells was negative. On EM, microvilli-like projections, junctional complexes, Golgi apparatus, and mucous granules were apparent in the majority of cells. Mitotic figures were observed by day 3 with Giemsa staining. Autoradiographically, these cells were able to incorporate [3H]TdR into the nuclei. Cells were capable of synthesizing DNA, and this function was inhibited by cycloheximide. Cells could be cultured for up to two weeks without fibroblast contamination. A method of primary monolayer culture of human gastric mucosa obtained by a routine endoscopic biopsy has been successfully developed.     

Interaction between prostatic fibroblast and epithelial cells in culture: role of androgen

We have established and characterized two cell lines from the ventral prostate gland of normal Nb rats: NbE-1 (prostatic epithelial) and NbF-1 (prostatic fibroblast) cell lines. To identify the direct mitogenic action of dihydrotestosterone (DHT), we incubated these cell lines alone and together (in the presence and absence of cell contact) with various concentrations of DHT (0.1-10,000 ng/ml) for 24-72 h and assayed for the rate of DNA synthesis and the total number of cells in tissue culture at specified time periods. Results demonstrate that the primary target for DHT mitogenic action is the prostatic fibroblasts. DHT inhibited the growth of prostatic epithelial cells by themselves, but stimulated prostatic epithelial cell growth when the epithelial cells were cocultured with prostatic fibroblasts. Furthermore, the cell-conditioned medium obtained from either the fibroblast or the epithelial cells stimulated in an autocrine or a paracrine manner the growth of prostatic cells in culture. These results are consistent with the concept that DHT stimulates the growth of prostatic epithelial cells indirectly via its direct mitogenic action on the prostatic fibroblasts. Because epithelial cells are the cell type principally responsible for converting testosterone to DHT, and the fibroblasts respond to the mitogenic action of DHT, our results support the concept that tight metabolic cooperation exists between prostatic epithelial and fibroblast cells. These data are in agreement with previous in vivo studies in which we have demonstrated that androgen receptors in the mesenchyme are obligatory for androgen-induced prostate growth and development.     

Primary culture of porcine pancreatic acinar cells

INTRODUCTION: The methodology of acinar cell culture has become of primary importance in the research of pancreatic physiology and pharmacology. AIM: To develop a method for primary culture of porcine pancreatic acinar cells. METHODOLOGY: Dispersed pancreatic acinar cells were made by RPMI-1640 medium containing collagenase III. After purification, the isolated acinar cells were cultured in RPMI-1640 medium with 2.5% fetal bovine serum. The morphologic characteristics of acinar cells were described. (3)H-thymidine incorporation of acinar cells and activity of amylase or lipase were determined during the culture. RESULTS: There were no remarkable morphologic changes in the pancreatic acinar cells during the 20-day culture. The acini showed the tendency of gathering but did not attach to the walls of the culture disks. Incorporation of (3)H-thymidine in acinar cells in the primary culture was well kept. The secretion of amylase or lipase from acini decreased with the time of culture. CONCLUSIONS: In the primary culture of acinar cells from porcine pancreas developed in this study, the acinar cells retained normal morphology and ability of growth but not secretion of amylase or lipase. The method would be beneficial for further experiments on acini of porcine pancreas.     

Defined culture conditions of human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into any tissue in the human body; therefore, they are a valuable resource for regenerative medicine, drug screening, and developmental studies. However, the clinical application of hESCs is hampered by the difficulties of eliminating animal products in the culture medium and/or the complexity of conditions required to support hESC growth. We have developed a simple medium [termed hESC Cocktail (HESCO)] containing basic fibroblast growth factor, Wnt3a, April (a proliferation-inducing ligand)/BAFF (B cell-activating factor belonging to TNF), albumin, cholesterol, insulin, and transferrin, which is sufficient for hESC self-renewal and proliferation. Cells grown in HESCO were maintained in an undifferentiated state as determined by using six different stem cell markers, and their genomic integrity was confirmed by karyotyping. Cells cultured in HESCO readily form embryoid bodies in tissue culture and teratomas in mice. In both cases, the cells differentiated into each of the three cell lineages, ectoderm, endoderm, and mesoderm, indicating that they maintained their pluripotency. The use of a minimal medium sufficient for hESC growth is expected to greatly facilitate clinical application and developmental studies of hESCs.     

Modifications of doubling potential of cultured human diploid cells by ionizing radiation and hydrocortisone

Cells possessing the ability to multiply into colonies have advanced successively to the next colony formation, which makes it possible to estimate the maximum doubling potential in the human diploid cell population. Attempts were made to modify the population doubling potential and the maximum doubling potential of human diploid cells with external agents. X-ray irradiation at high dose rates reduced the population doubling potential (Ban et al., 1980), while hydrocortisone had the advantage of extending the population life span, as has neen reported. The magnitude of extension acquired was equivalent to about a quarter of the non-treated population's life span.

However, X-irradiation and hydrocortisone treatment failed to change the maximum doubling potential. This means that ionizing radiation might accelerate the progression of cells to non-cycling state, while hydrocortisone might decelerate it. Our results support the hypothesis that the growth and death of cultured human diploid cells might reflect cell differentiation in vitro (Bell et al., 1978).     

Hepatocyte growth factor is required for progestin-induced epithelial cell proliferation and alveolar-like morphogenesis in serum-free culture of normal mammary epithelial cells

The steroid hormones, estrogen and progesterone, are required for mammary epithelial cell proliferation and alveolar morphogenesis in vivo. We have developed a minimally supplemented, serum-free medium, collagen gel primary mammary culture system to determine the mechanism of progestin-induced proliferation and alveolar morphogenesis. In epithelial cells cultured alone, treatment with progestin (R5020) alone produced a lumen within the epithelial organoids, but did not stimulate epithelial cell proliferation. The formation of lumens was associated with increased apoptosis, targeted within the organoids. We have previously reported that in our culture system hepatocyte growth factor (HGF) increases epithelial cell proliferation and induces a tubulo-ductal morphological response. In the present report we show that treatment with HGF and progestin (R5020) further increases epithelial proliferation above that with HGF alone and also produces an alveolar-like morphology similar to that observed in vivo in response to progestin treatment. To the best of our knowledge this is the first in vitro demonstration of both progestin-induced proliferation and alveolar-like morphogenesis of normal nonpregnant mouse mammary epithelial cells in vitro. These results suggest that HGF may play a crucial role in progestin-induced proliferation and morphogenesis in vivo.     

Effect of Interlukin-1β on proliferation of gastric epithelial cells in culture

Background  

Helicobacter pylori is the main risk factor for the development of non-cardia gastric cancer. Increased proliferation of the gastric mucosa is a feature of H. pylori infection. Mucosal interkeukin-1β production is increased in H. pylori infection and IL-1β genotypes associated with increased pro-inflammatory activity are risk factors for the development of gastric cancer. The effect of IL-1β on gastric epithelial cell proliferation has been examined in this study.