Role of biotin-containing membranes and nuclear distribution in differentiating human endometrial cells

Human Ishikawa endometrial cells form domes when confluent monolayers are stimulated with fresh fetal bovine serum. Extensive structural and biochemical changes have been detected during the approximately 30 h differentiation period. The earliest detectable change involves the formation of multinucleated structures and the appearance of “granules” that stain for biotin within those structures. Nuclei become associated with each other and are ultimately enclosed within a biotin-containing membrane. Aggregated membrane-sheathed nuclei and the cells containing them begin to elevate from the dish as biotin staining becomes apparent in apical membranes. The elevated structures are called predomes and consist of one or more very large cells containing the sheathed nuclei. Apical membranes of these unusual cells extend far out into the medium in structures that resemble endometrial pinopods. A lumen under the elevated cells fills with transcytosed fluid. As differentiation proceeds, highly concentrated chromatin material that was flattened against apical and lateral membranes of the predome cells begins to disperse. Small mononuclear cells evolve from larger predome cells. Apical membranes of predome and dome cells continue to stain for biotin. Gel electrophoresis of SDS-solubilized biotin-containing membranes, followed by Western blot analysis using avidin-linked peroxidase, resulted in three stained bands with molecular weights similar to those of the mitochondrial carboxylases: propionyl carboxylase, methylmalonyl carboxylase, and pyruvate carboxylase. J. Cell. Biochem. 71:400–415, 1998. © 1998 Wiley-Liss, Inc.     

Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture

Smooth muscle cell (SMC) plays critical roles in many human diseases, an in vitro system that recapitulates human SMC differentiation would be invaluable for exploring molecular mechanisms leading to the human diseases. We report a directed and highly efficient SMC differentiation system by treating the monolayer-cultivated human embryonic stem cells (hESCs) with all-trans retinoid acid (atRA). When the hESCs were cultivated in differentiation medium containing 10microM RA, more than 93% of the cells expressed SMC-marker genes along with the steadily accumulation of such SMC-specific proteins as SM alpha-actin and SM-MHC. The fully differentiated SMCs were stable in phenotype and capable of contraction. This inducible and highly efficient in vitro human SMC system could be an important resource to study the mechanisms of SMC phenotype determination in human.     

Gland formation from human endometrial epithelial cells in vitro

Summary We have developed methods for the culture of human endometrial glandular epithelia in vitro. The culture medium is serum-free and is used in combination with Matrigel, an extracellular matrix material applied as a coating on cell culture plates. Cell growth begins as a monolayer, but the cells subsequently form glandular or organoid structures. The glands are composed of polar columnar cells facing a central lumen, which is enclosed by the apical surfaces of cells displaying numerous microvilli and sealed by tight junction complexes. The ability to study in vitro the complex process of glandular morphogenesis represents an important new tool in cell biology which may be used to investigate growth regulation, hormone production and dependency, and cellular recognition and interactions. Ultimately, these characteristics may be applied to study the alterations of glandular epithelia associated with neoplasia. This work was supported by NIH grants CA31733 and CA09156 and NIEHS contract ES55092     

Ultrastructure of human endometrial epithelium in monolayer culture with and without steroid hormones

Summary Colonies of cells of epithelioid appearance were identified in monolayer cultures grown up to 50 days from normal human endometrial cell suspensions obtained by a method designed to insure a maximum harvest of glandular cells. Groups of these cells were separated from stromal cells by means of cloning cylinders. Studies comparing the ultrastructure of cells of this type to fresh endometrial tissue revealed a number of similarities. The morphological characteristics common to both types of samples included junctional complexes, perinuclear microfilaments and microvilli with glycocalyx. Other common features were prominent nucleoli, well developed Golgi, rough endoplasmic reticulum and membranebound electron-dense bodies in the cytoplasm. A stripping technique applie to the fetal bovine serum used in the nutrient medium made it possible to initiate cultures in a steroidfree environment and to maintain them in the presence of the specified concentration of estradiol and/or progesterone. Isolation of epithelial cells of endometrium in monolayer culture may provide a useful model system in which to study the specific effects of steroid hormones on cellular function and differentiation. Supported by grants from the National Institutes of Health (CA 18678 and CA 07368).     

Development and retroviral transduction of porcine neonatal pancreatic islet cells in monolayer culture

To learn more about the potential of neonatal porcine pancreatic duct and islet cells for xenotransplantation, the development of these cells when cultured as monolayers was evaluated. Immunostaining for islet hormones and cytokeratin-7 revealed that day eight monolayers consisted of approximately 70% duct cells and less than 10% beta cells. Using Ki-67 immunostaining as a proliferation marker, the fraction of beta cells in the cell cycle was shown to decrease from 20% at day three to 10% at day eight, and for duct cells from 36 to 19%. Insulin secretion increased 2.4-fold upon glucose stimulation, and 38-fold when 10 mm theophylline was added, showing the responsiveness of the neonatal beta cells. Reaggregated monolayers consisted mostly of duct cells, but 4 weeks after transplantation, grafts contained predominantly endocrine cells, with duct cells being almost absent, suggesting in vivo differentiation of duct cells to endocrine cells. Monolayer susceptibility to retroviral transduction was also investigated using a Moloney Murine Leukemia Virus-based vector. Approximately 60% of duct cells but less than 5% of beta cells expressed the transgene, indicating that precursor duct cells are better targets for transgene expression. These results show that porcine neonatal pancreatic cells can be cultured as monolayers in preparation for transplantation. Furthermore, in such a culture setting, precursor duct cells have a high rate of proliferation and are more efficiently transduced with a retrovirus-based reporter gene than are beta cells.     

Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells

The potential of mesenchymal stem cells (MSC) to differentiate into different cell types has opened up the possibility of using these cells clinically to treat a variety of disorders. In this study we describe the use of human umbilical cord blood serum (CBS) as a replacement for fetal bovine serum (FBS) for culturing MSC from different sources. MSC from human and swine bone marrow and human umbilical cord blood were cultured in the presence of DMEM/F12 containing either FBS or CBS. Human MSC cultured in presence of FBS or CBS showed typical fibroblast-like morphology, which is characteristic of MSC. 99% of the cells cultured in FBS had a CD73+/CD105+/CD45- phenotype compared to 96% of cells cultured in CBS. Cells cultured in CBS had a significantly higher cell count as compared to cells cultured in FBS. Swine Bone Marrow MSC cultured in the presence of FBS and CBS were morphologically and phenotypically similar. Human umbilical cord blood serum supports the growth of MSC. While no significant differences were observed in the MSC numbers in swine cells cultured in the presence of FBS or CBS, human cells showed a greater proliferation potential in the presence of CBS as compared to FBS. Therefore, CBS can be used as an effective substitute to FBS for developing clinically useful protocols for culturing MSC.     

β-ME和BHA体外诱导人胎肝CD34+细胞向神经组织细胞分化研究

目的:建立巯基乙醇(β-mercaptoethanol,β-ME)和丁羟回醚(butylated hydroxyanisole,BHA)体外诱导人胎肝(fetal liver,FL)干细胞向神经细胞分化模型.方法:采用MACS试剂盒分离人胚胎肝CD34 细胞,以DMEM 10%胎牛血清培养液培养;第五代细胞待细胞融合达80%后,用DMEM 10%胎牛血清 1mmol/L β-ME 0.2 mmol/L BHA诱导24 h,PBS洗涤.然后在无血清培养基中培养5 h～5 d.用免疫细胞化学方法分析诱导前后的细胞表型特点.结果:经β-ME BHA诱导处理后,细胞表现神经元样细胞形态,表达神经组织细胞特异蛋白,如neustin、NeuN、NF-M、TuJ-1和NSE.统计显示81%细胞NeuN染色阳性,75%细胞TuJ-1染色阳性,47%染色NF-M阳性,90%染色NSE阳性.结论:β-ME和BHA能够诱导体外培养的人FL CD34 细胞分化为具有神经细胞特异性抗原和成分的神经样细胞;胚胎肝细胞具有向神经组织分化的潜能.     

Biotransformation of tamoxifen in a human endometrial explant culture model

Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium.     

Serum-free culture of stromal and functionally polarized epithelial cells of guinea-pig endometrium: a potential model for the study of epithelial-stromal paracrine interactions.

Stromal and glandular epithelial (GE) cells were isolated from guinea-pig endometrium and growth to near confluency (6-8 days) in primary culture on plastic surfaces in a serum-supplemented medium (SSM). The stromal cells were subcultured on plastic dishes and maintained for 72 h in SSM. Then SSM was replaced by a chemically defined medium (CDM) and the stromal cells grown to confluency (5-7 days). The GE cells were subcultured in CDM, on a basement membrane matrix (Matrigel) applied to permeable Millicell-PC filters, and grown to confluency (5 days). Homogeneity of the subcultured endometrial cell populations was ascertained immunocytochemically. The filter-cultured GE monolayers were polarized morphologically, and displayed epithelial-specific specialized structures. These monolayers had functional tight junctions as verified by a measurable transepithelial resistance. The subcultured cell populations were distinguished by an analysis of their cellular and secretory proteins after labelling with [35S]-methionine and analysis by polyacrylamide gel electrophoresis. The filter-cultured GE monolayers allowed identification of the proteins released vectorially in the apical or the basal secretory compartment, thus demonstrating the functional polarization of GE cells in this bicameral culture system. Within the defined conditions of this culture system, the paracrine factors released by the two endometrial cell populations as well as the interplay of stromal-epithelial interactions and ovarian hormones could be investigated.     

Growth-inhibitory effect of TGF-B on human fetal adrenal cells in primary monolayer culture

We examined the effects of transforming-growth factor-B (TGF-B) on growth ([3H]-thymidine uptake) and function (dehydroepiandrosterone sulfate [DHAS] and cortisol production) of human fetal zone adrenal cells. Results indicate that TGF-B significantly inhibits, in a dose-related manner, both basal and epidermal growth factor (EGF)-stimulated cell growth: IC50 = 0.1-0.25 ng/ml. EGF is ineffective in overcoming the inhibitory effect of TGF-B, suggesting a noncompetitive antagonism between the two factors. Also, the inhibitory effect of TGF-B is additive to that of adrenocorticotropic hormone (ACTH). On the other hand, TGF-B (1 ng/ml) does not significantly change basal or ACTH-stimulated DHAS or cortisol secretion. We conclude that, unlike its effect on other steroid-producing cells, TGF-B inhibits growth of fetal zone cells and does not appear to have a significant inhibitory effect on steroidogenesis.     

Oxygen consumption of human heart cells in monolayer culture

Tissue engineering in cardiovascular regenerative therapy requires the development of an efficient oxygen supply system for cell cultures. However, there are few studies which have examined human cardiomyocytes in terms of oxygen consumption and metabolism in culture. We developed an oxygen measurement system equipped with an oxygen microelectrode sensor and estimated the oxygen consumption rates (OCRs) by using the oxygen concentration profiles in culture medium. The heart is largely made up of cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. Therefore, we measured the oxygen consumption of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), cardiac fibroblasts, human cardiac microvascular endothelial cell and aortic smooth muscle cells. Then we made correlations with their metabolisms. In hiPSC-CMs, the value of the OCR was 0.71 ± 0.38 pmol/h/cell, whereas the glucose consumption rate and lactate production rate were 0.77 ± 0.32 pmol/h/cell and 1.61 ± 0.70 pmol/h/cell, respectively. These values differed significantly from those of the other cells in human heart. The metabolism of the cells that constitute human heart showed the molar ratio of lactate production to glucose consumption (L/G ratio) that ranged between 1.97 and 2.2. Although the energy metabolism in adult heart in vivo is reported to be aerobic, our data demonstrated a dominance of anaerobic glycolysis in an in vitro environment. With our measuring system, we clearly showed the differences in the metabolism of cells between in vivo and in vitro monolayer culture. Our results regarding cell OCRs and metabolism may be useful for future tissue engineering of human heart.     

牛雄性生殖系干细胞的体外诱导分化

雄性生殖系干细胞(Male germ-line stem cells, mGSCs)是一群具有高度自我更新能力和分化潜能的细胞, 是雄性成体内唯一可复制的二倍体永生细胞。转基因技术与雄性生殖系干细胞异体及异种移植技术相结合, 将会为克隆动物、转基因动物生产及一些人类遗传性疾病的基因治疗提供新的机遇与途径。本试验采用组合酶消化和选择贴壁法, 对5月龄、6月龄牛胎儿及新生牛雄性生殖系干细胞体外培养及分化进行了研究。试验结果显示, 睾丸支持细胞对雄性生殖系干细胞体外增殖、分化所必需的, 同时对数期睾丸支持细胞对雄性生殖系干细胞贴壁、增殖与分化效果明显; 共培养16 d后, 牛雄性生殖系干细胞分化为长形精子细胞, 试验建立了牛雄性生殖系干细胞体外诱导培养分化体系。     

Nitric oxide induces segregation of decay accelerating factor (DAF or CD55) from the membrane lipid-rafts and its internalization in human endometrial cells

Recent studies suggest that DAF (decay accelerating factor), a complement regulatory protein, present in lipid rafts, is utilized by Dr fimbriated Escherichia coli for their binding and internalization. Previous studies in our laboratory have shown that NO (nitric oxide) can reduce the invasion of Dr(+) E. coli and the severity of uterine infection in pregnant rats. Also, the expression level of DAF both at the mRNA and protein levels has been shown to be reduced by NO. Therefore NO mediated down-regulation of DAF appears to be an important factor in reducing the susceptibility to E. coli infection. However, it is unclear if NO can actually modulate the membrane association of DAF and therefore initial bacterial binding to cells. We found that NO induces the delocalization of DAF from the GM1-rich lipid rafts. Using biochemical and cell biological approaches in a uterine epithelial cell model (Ishikawa cells), DAF accumulates in caveolae upon exposure to NO. Interaction of DAF with the caveolar protein, caveolin1, leads to their internalization by endosomes. NO-induced delocalization of DAF from the lipid raft and its accumulation in caveolae are mediated through a cGMP (cyclic guanosine monophosphate) pathway. The acute localized synthesis of NO and its influence on DAF localization may represent an important unrecognized phenomenon of host defence against Dr(+) E. coli bacteria, as well as many disease conditions that involve complement system.     

Effects of culture conditions on taurine uptake by various variants of human endometrial carcinoma cells in culture

Summary. The general properties of the taurine uptake in human endometrial tumoral Ishikawa cells were similar to those usually found in other tissues. Uptake was notably affected by the oxygen pressure, being higher at the physiological pO₂ of the endometrium (40mm Hg, equivalent to 5% O₂) compared to that used under standard experimental culture conditions (160mm Hg or 20% O₂). Uptake of taurine was also density-dependent in Ishikawa cells and was significantly decreased at confluence. Uptake regulation by PKC driven phosphorylation occurs only in growing cells and not in resting cells. The taurine uptake of three Ishikawa cell lines was very different. The taurine uptake of one of the cell lines was affected by estradiol, probably through a non-genomic pathway, whereas tamoxifen had no effect in all cell lines.     

Reaggregation of fetal rat brain cells in a stationary culture system I: Methodology and cell identification

Summary A stationary tissue culture system for reaggregation cultures of rat brain cells is described. Aggregates were formed by placing cells at high concentrations in liquid overlay cultures on a nonadherent nutrient agar surface. No physical stress in the form of rotation or shaking was applied to the aggregating cell population. Transmission electron microscopy and immunohistochemistry showed that the cells developed from homogeneously dispersed, immature cells in Day 4 aggregates, to mature astrocytes, oligodendrocytes, and neurons in Day 20 aggregates. Twenty days and older aggregates had a tightly packed neuropil which was most prominent in a cell-sparse outer layer of the aggregates. When the aggregates were allowed to adhere to a substrate, both glial fibrillary acidic protein (GFAP) positive and negative cells were observed migrating out from the aggregates. Cells giving a positive reaction for neuron specific enolase (NSE) were also present. This reaggregation procedure, with transfer of selected brain cell aggregates into agar-coated multiwells is an alternative three-dimensional culture system which can be potentially useful in the study of morphogenesis and cell interactions in the nervous system. This project was supported by the Norwegian Cancer Society.     

Expression and action of hepatocyte growth factor in bovine endometrial stromal and epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that acts on various epithelial cells. The objectives of this study were to determine whether HGF altered the proliferation and prostaglandin (PG) secretion of bovine endometrial stromal and epithelial cells in vitro. We also observed HGF and HGF receptor (c-met) mRNA expression in cultured bovine endometrial stromal and epithelial cells by RT-PCR. Stromal and epithelial cells obtained from cows in early stage of the estrous cycle (days 2-5) were cultured in DMEM/Ham's F-12 supplemented with 10% calf serum. The cells were exposed to HGF (0-10 ng/ml) for 2, 4, or 6 days. HGF significantly increased the total DNA in epithelial (P < 0.05), but not stromal cells. In another experiment, when the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA containing HGF 0-100 ng/ml and the cells were cultured for 24 hr. The HGF stimulated PGF2alpha secretion in epithelial, but not stromal cells. RT-PCR revealed that mRNA of HGF is expressed only in stromal cells, and that c-met mRNA is expressed in both stromal and epithelial cells. These results suggest that HGF plays roles in the proliferation and the regulation of secretory function of bovine endometrial epithelial cells in a paracrine fashion.