Androstenedione metabolism in human lung fibroblasts

Human lung fibroblasts in culture metabolized [3H]androstenedione to a number of different compounds, including testosterone, 5 alpha-androstanedione, androsterone, 5 alpha-dihydrotestosterone, isoandrosterone, and 5 alpha-androstane-3 alpha,-17 beta-diol. The major products were 5 alpha-androstanedione and testosterone. Estrone, estradiol-17 beta and 5 beta-reduced steroids were not formed. The production rates of testosterone and 5 alpha-androstanedione from [3H]androstenedione by lung fibroblasts were studied both as a function of incubation time and substrate concentration. The rates of formation of testosterone and 5 alpha-androstanedione remained linear with time up to 4 h. The apparent Km of human lung fibroblast 5 alpha-reductase was 1 microM, and that of 17 beta-hydroxysteroid oxidoreductase was 11 microM. The findings of this study suggest that mesenchyma may contribute to the metabolism of androstenedione in human lung tissue.     

Serial culturing of human bronchial epithelial cells derived from biopsies

Summary In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.     

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells

BackgroundIntravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.MethodsHuman AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.ResultsHSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen–G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.ConclusionsHuman AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.     

p53 mutations in tumors derived from irradiated human thyroid epithelial cells

A non-tumorigenic human thyroid epithelial cell line (HTori-3) has been transformed into tumorigenic cells by exposure in vitro to alpha particles or gamma-radiation. These transformants were tumorigenic in athymic nude mice and tumors were transplantable into other nude mice. To further characterize processes involved in neoplastic progression, the tumor cell lines derived from these radiation-induced primary tumors were screened for mutations in the p53 tumor suppressor gene. p53 mutation was detected by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 inclusive. Mutations detected by SSCP analysis were confirmed by sequencing. Mutations were detected in all four exons analysed, although there was no correlation between dose, LET or mutation position or frequency. Mutations in p53 exons 6 and 7 have been reported in the childhood papillary thyroid carcinomas in Belarus presumably as a result of radioiodine fall-out. Similarly here, p53 mutations are induced experimentally during the development of human thyroid tumors generated by irradiation of a human thyroid epithelial cell line in vitro.     

The epithelial cells and cell fragments in human milk

Summary The cell fragments and epithelial cells in human milk were examined in samples obtained from 30 women: 3 of these provided sequential samples at weekly intervals for 110 days. Membrane-bound cytoplasmic fragments in the sedimentation pellet greatly outnumbered the population of intact cells in all samples. Most of the fragments were derived from secretory cells and contained numerous cisternae of rough endoplasmic reticulum, lipid droplets and Golgi vesicles containing casein micelles. Secretory epithelial cells were present in small numbers in all samples and after the 2nd month of lactation replaced the macrophage as the predominant cell type. Ductal epithelial cells represented less than 1% of the total cell population up to 8 days post-partum, but thereafter they were very rarely found. They occurred in aggregates of 2–4 cells and possessed tight junctions that circumscribed the area of cell-cell contact. All samples of milk contained squamous epithelial cells derived from the galactophores and/or the skin of the nipple. Bacteria were often attached to the surface of the squamous cells. The possible relationship between the presence of secretory epithelial cells in milk and the occurrence of milk proteins in the blood of lactating women is discussed.     

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from The Polycystic Kidney Foundation. We gratefully acknowledge the support of the Children’s Medical Research Institute and Children’s Miracle Network Foundation.     

Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75^NTR), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75^NTR was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75^NTR was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75^NTR is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.     

A simple culture method for epithelial stem cells derived from human hair follicle

The challenge arises among researchers when hair follicle stem cells (HFSCs) derived from a human hair follicle remain poorly expanded in defined culture medium. In this study, we isolated the HFSCs and they became confluent after 10 days of cultivation. Comparing the viability of HFSCs cultured in defined keratinocytes serum free medium (KSFM) in a coated plate and CnT07 medium in an uncoated plate, the number of HFSCs cultured in CnT07 was significantly higher at days 2, 4, 6 and 8 (P=0.004). The population doubling time of HFSCs was 21.48±0.44 hours in non-coated plates with CnT07 and 30.73±0.75 hours in coated plates with KSFM. Our primary HFSC cultures were positive for CD200 and K15 with brownish color. Flow cytometry analysis showed that the percentage of HFSCs expressing CD200 and K15 were 65.20±3.16 and 72.07±6.62 respectively. After reaching 100% confluence, the HFSCs were differentiated into an epidermal layer in vitro using CnT02-3D defined media. HFSCs were differentiated into an epidermal layer after 2 weeks of induction. Involucrin- and K6-positive cells were detected in the differentiated epidermal layer. This method is a simple technique for HFSC isolation and has a lower cost of processing and labor, and it represents a promising tool for skin tissue engineering.     

Androstenedione metabolism in human uterine tissues: Endometrium,myometrium and leiomyoma

Androstenedione was metabolized in vitro by human endometrium, myometrium and leiomyoma, to its 5α-reduced metabolites: 5α-androstan-3,17-dione (5α-androstanedione) and androsterone as well as to testosterone, 17β-hydroxy-5α-androstan-2-one(5α-DHT) and 5α-androstan-3α, 17β-diol (3α-diol). Uterine tissue showed a similar enzymatic profile to the androgen responsive tissues; these data suggest that androgens may have a functional role in the uterine pathophysiology.     

Regenerated luminal epithelial cells are derived from preexisting luminal epithelial cells in adult mouse prostate

Determining the source of regenerated luminal epithelial cells in the adult prostate during androgen deprivation and replacement will provide insights into the origin of prostate cancer cells and their fate during androgen deprivation therapy. Prostate stem cells in the epithelial layer have been suggested to give rise to luminal epithelium. However, the extent of stem cell participation to prostate regrowth is not clear. In this report, using prostate-specific antigen-CreER(T2)-based genetic lineage marking/tracing in mice, preexisting luminal epithelial cells were shown to be a source of regenerated luminal epithelial cells in the adult prostate. Prostatic luminal epithelial cells could survive androgen deprivation and were capable of proliferating upon androgen replacement. Prostate cancer cells, typically exhibiting a luminal epithelial phenotype, may retain this intrinsic capability to survive and regenerate in response to changes in androgen signaling, providing part of the mechanism for the ultimate failure of androgen deprivation therapy in prostate cancer.     

Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation

Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.     

Cloned kids derived from caprine mammary gland epithelial cells

The use of nucleus transfer techniques to generate transgenic dairy goats capable of producing recombinant therapeutic proteins in milk could have a major impact on the pharmaceutical industry. However, transfection or gene targeting of nucleus transfer donor cells requires a long in vitro culture period and the selection of marker genes. In the current study, we evaluated the potential for using caprine mammary gland epithelial cells (CMGECs), isolated from udders of lactating F1 hybrid goats (Capra hircus) and cryopreserved at Passages 24 to 26, for nucleus transfer into enucleated in vivo-matured oocytes. Pronuclear-stage reconstructed embryos were transferred into the oviducts of 31 recipient goats. Twenty-three (74%), 21 (72%), and 14 (48%) recipients were confirmed pregnant by ultrasonography on Days 30, 60, and 90, respectively. Four recipients aborted between 35 and 137 d of gestation. Five recipients carried the pregnancies to term and delivered one goat kid each, one of which subsequently died due to respiratory difficulties. The remaining four goat kids were healthy and well. Single-strand conformation polymorphism analysis confirmed that all kids were clones of the donor cells. In conclusion, the CMGECs remained totipotent for nucleus transfer.     

Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×10? RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.     

Essential fatty acid metabolism in cultured human airway epithelial cells.

To characterize essential fatty acid metabolism of human airway epithelium, we examined the capacity of epithelial cells to incorporate and desaturate/elongate 18:2(n - 6) and the turnover of phospholipid fatty acyl chains in these cells. Epithelial cells were cultured for 5-7 days and incubated with [1-14C]18:2(n - 6) (1 microCi, 100 nmol). The essential fatty acid profile of the cells was readily modified by 18:2(n - 6) supplementation to culture medium. After 4 h incubation, 32 +/- 5.6 nmol of [1-14C]18:2(n - 6) was incorporated into phospholipids (65 +/- 9.5%, of which 74% was incorporated into phosphatidylcholine (PC)) and neutral lipid (31 +/- 10%) per mg protein of cultured cells. 30 +/- 8% of [1-14C]18:2(n - 6) incorporated, was converted to homologous trienes, tetraenes and pentaenes, the major products being 20:3(n - 6) and 20:4(n - 6). The conversion of 18:2(n - 6) was time-dependent and donor age-related. A higher proportion of 20:3(n - 6) and 20:4(n - 6) was incorporated into phosphatidylinositol (PI) and phosphatidylethanolamine (PE). About 10-15% of total products formed from 18:2(n - 6) was released from membrane to culture medium. Both 20:4(n - 6) and 20:5(n - 3) inhibited 18:2(n - 6) incorporation and desaturation. Rate of incorporation of 18:2(n - 6) was more than either 18:1(n - 9) or 16:0. With pulse-chase studies, the half-life of 18:2(n - 6) in PC, PI and PE was estimated to be 5.5, 6.0 and 7.3 h, respectively. These data indicate active metabolism of essential fatty acids in human airway epithelial cells. This metabolism may play a key role in the regulation of membrane properties and function in these cells.     

Characterization of epithelial cell cultures derived from human tracheal glands

Summary Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 μg/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 μg/ml), and bovine pituitary extract (25 μg/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide β-d-galactose-(l→ 3)N-acetyld-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl⁻ channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established. This research was supported by USPHS grants HL 41979 and HL 33142 from the National Heart, Lung and Blood Institutes.