Appearance and endocytic activity of epithelial cells from human efferent ducts in primary monolayer culture

Summary The culture of epithelial cells lining human efferent ducts, obtained from prostatic carcinoma patients, is described. Ciliated cells were observed to beat for at least one month on plastic. On pervious filters low cuboidal cells characterized the monolayers. Cells comprising monolayers over the filter were 5 to 9 m in height whereas taller cells were found over the original fragments (14 m). Some non-ciliated cells contained dark and light vacuoles, others were found to lack them. Both non-ciliated and ciliated cells maintained tight junctional complexes restricting the paracellular movement of horseradish peroxidase. Both types of cultured cells exhibited fluid-phase and adsorptive endocytosis from both apical and basal surfaces. It is reported for the first time that the monolayers form high resistance barriers (150 cm²) that prevent the apical medium from draining to the basal compartment over 24 h.     

Identification of novel epithelial stem cell-like cells in human deciduous dental pulp

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, ΔNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.     

Altered morphology in cultured rat intestinal epithelial IEC-6 cells is associated with alkaline phosphatase expression

Non-transformed, rat intestinal epithelial cells (IEC-6), and human intestinal colonic carcinoma cells (CACO-2) have both been used to study processes of epithelial cell differentiation. However, only CACO-2 cells have been described as spontaneously expressing phenotypic changes of differentiation in culture. We report here that when IEC-6 cells are grown in post-confluent culture, they develop structural changes similar to those seen in cells induced to differentiate by culture on Englebreth-Holm-Swarm (EHS) extracellular matrix proteins. Correlated with this morphological change is loss of nuclear localization of c-myc protein and development of cell surface alkaline phosphatase (ALP) enzymatic activity. Messenger RNAs for liver and intestinal isoforms of ALP were expressed in both pre- and post-confluent cells. Inhibition of ALP activity in post-confluent cells by levamisole indicated the expressed ALP activity to be of the liver isoform. We suggest the expression of ALP activity, which occurs concomitantly with morphological alterations in post-confluent IEC-6 cells, represents increased expression and localization to the cell surface of the liver isoform of ALP. Cultured IEC-6 cells may provide a non-transformed, in vitro alternative to CACO-2 cells for study of epithelial cell differentiation.     

Effect of transferrin on alkaline phosphatase activity and collagen synthesis in osteoblastic cells derived from newborn mouse calvaria

The effect of transferrin was tested on osteoblastic cells (clone MC3T3-E1) cultured in serum-free medium containing 1% bovine serum albumin (BSA). Transferrin (Tf) stimulated increases of protein content and protein synthesis, but not of DNA content and cell number, in the cells. This protein also increased alkaline phosphatase activity and collagen synthesis in combination with 1% BSA. Actinomycin D and cycloheximide inhibited alkaline phosphatase activity induced by Tf, suggesting that Tf may enhance de novo synthesis of the enzyme. These results indicate that Tf may be involved in differentiation of osteoblastic cells, but not in their proliferation, in vitro.     

Continuous multiplication of rabbit tracheal epithelial cells in a defined,hormone-supplemented medium

Summary An improved Ham’s F12 nutrient medium supplemented with epidermal growth factor (EGF), insulin (INS), and transferrin (TF) was developed for continuous proliferation and clonal growth of primary rabbit tracheal epithelial (TE) cells in culture. The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measurable stimulation on TE cell growth and plating efficiency. However, serum levels higher than 0.1% inhibited cell growth and also masked the growth stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal growth occurred at a seeding density of 17 cells/cm² with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a 0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture for extended periods of time without the aid of a fibroblast feeder layer or explant tissue. This system could be useful for the study of cell differentiation of tracheal epithelium.     

Age-related changes in mesenchymal stem cells derived from rhesus macaque bone marrow

The regeneration potential of mesenchymal stem cells (MSCs) diminishes with advanced age and this diminished potential is associated with changes in cellular functions. This study compared MSCs isolated from the bone marrow of rhesus monkeys (rBMSCs) in three age groups: young (< 5 years), middle (8-10 years), and old (> 12 years). The effects of aging on stem cell properties and indicators of stem cell fitness such as proliferation, differentiation, circadian rhythms, stress response proteins, miRNA expression, and global histone modifications in rBMSCs were analyzed. rBMSCs demonstrated decreased capacities for proliferation and differentiation as a function of age. The production of heat shock protein 70 (HSP70) and heat shock factor 1 (HSF1) were also reduced with increasing age. The level of a core circadian protein, Rev-erb α, was significantly increased in rBMSCs from old animals. Furthermore, analysis of miRNA expression profiles revealed an up-regulation of mir-766 and mir-558 and a down-regulation of mir-let-7f, mir-125b, mir-222, mir-199-3p, mir-23a, and mir-221 in old rBMSCs compare to young rBMSCs. However, there were no significant age-related changes in the global histone modification profiles of the four histone core proteins: H2A, H2B, H3, and H4 on rBMSCs. These changes represent novel insights into the aging process and could have implications regarding the potential for autologous stem cells therapy in older patients.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Biological characteristics of foam cell formation in smooth muscle cells derived from bone marrow stem cells

Bone marrow mesenchymal stem cells (BMSC) can differentiate into diverse cell types, including adipogenic, osteogenic, chondrogenic and myogenic lineages. There are lots of BMSC accumulated in atherosclerosis vessels and differentiate into VSMC. However, it is unclear whether VSMC originated from BMSC (BMSC-SMC) could remodel the vessel in new tunica intima or promote the pathogenesis of atherosclerosis. In this study, BMSC were differentiated into VSMC in response to the transforming growth factor β (TGF-β) and shown to express a number of VSMC markers, such as α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain1 (SM-MHC1). BMSC-SMC became foam cells after treatment with 80 mg/L ox-LDL for 72 hours. Ox-LDL could upregulate scavenger receptor class A (SR-A) but downregulate the ATP-binding cassette transporter A1 (ABCA1) and caveolin-1 protein expression, suggesting that modulating relative protein activity contributes to smooth muscle foam cell formation in BMSC-SMC. Furthermore, we found that BMSC-SMC have some biological characteristics that are similar to VSMC, such as the ability of proliferation and secretion of extracellular matrix, but, at the same time, retain some biological characteristics of BMSC, such as a high level of migration. These results suggest that BMSC-SMC could be induced to foam cells and be involved in the development of atherosclerosis.     

Optimization of primary culture condition for mesenchymal stem cells derived from umbilical cord blood with factorial design

Mesenchymal stem cells (MSCs) can not only support the expansion of hematopoietic stem cells in vitro, but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However, it proved challenging to culture MSCs from umbilical cord blood (UCB) with a success rate of 20–30%. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In this work, fractional factorial design was applied to study the effect of cell inoculated density, combination and dose of cytokines, and presence of serum and stromal cells. The cultured UCB‐MSC‐like cells were characterized by flow cytometry and their multilineage differentiation potentials were tested. The optimal protocol was identified achieving above 90% successful outcome: 2 × 10⁶ cells/mL mononuclear cells inoculated in Iscove's modified Dulbecco's medium supplied with 10% FBS, 15 ng/mL IL‐3, and 5 ng/mL Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Moreover, the UCB‐MSC‐like cells expressed MSC surface markers of CD13, CD29, CD105, CD166, and CD44 positively, and CD34, CD45, and human leukocyte antigens‐DR (HLA‐DR) negatively. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes, and adipocytes similarly to MSCs derived from bone marrow. In conclusion, we have developed an efficient protocol for the primary culture of UCB‐MSCs by adding suitable cytokines into the culture system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Reduction of chemokine IL-8 and RANTES expression in human bronchial epithelial cells by a sea-water derived saline through inhibited nuclear factor-kappaB activation

The NaCl content of airway surface fluid is believed to be of central importance in lung pathology. To test whether the Na+ concentration could influence the inflammatory response in human bronchial epithelial cells (BECs), we investigated the interleukin (IL)-8 and RANTES expression in BECs exposed to an isotonic sea-water derived low Na+ (ISW) saline compared to isotonic 0.9% NaCl saline. Exposure of BECs to ISW saline caused a significant decrease in IL-8 and RANTES gene expression and protein production as compared to that observed with 0.9% NaCl saline. Furthermore, we observed a concomitant reduction of phosphorylated IkappaBalpha associated with a marked inhibition of NF-kappaB-DNA binding activity in BECs exposed to ISW saline as compared to 0.9% NaCl saline. These findings support a new role for Na+ in the pathogenesis of airway inflammatory disorders. Therapies targeted at lowering Na+ level in airway epithelium may be beneficial in treating inflammatory lung diseases.     

Regulation of cell proliferation in a stratified culture system of epithelial cells from prostate tissue

Mechanisms controlling epithelial proliferation and differentiation in the prostate have been primarily investigated in mouse models. The regulation of proliferation and differentiation is poorly understood in human prostate epithelial cells. In vivo, the glandular prostate epithelium consists of a p63-positive proliferating basal cell layer and a post-mitotic p27-positive secretory cell layer. We have established an organized stratified culture system of human primary prostate epithelial cells to gain insight into mechanisms regulating proliferation and differentiation. In this system, expression of p63 is observed in the bottom layer. In addition, BrdU incorporation persists even though cells are confluent. In contrast, in the upper layer, p63 expression is greatly diminished, p27 is expressed, and the cells are growth arrested. Overexpression of cyclin D1 or knockdown of p27 does not increase proliferation. After inactivation of the nuclear phosphoprotein Rb, the cell layers remain organized and cell proliferation increases only in the bottom layer. Furthermore, the expression of p63 remains confined to the bottom layer after Rb inactivation. Altogether, this in vitro model recapitulates certain aspects of in vivo growth regulation and differentiation and suggests that the loss of Rb family proteins in human cells trigger hyperplasia but is not sufficient for transformation.This work was supported by the Departments of Pathology and Urology at Weill Medial College, by grants DAMD-17-02-1-0159, MEDC-GR-355, and P30 CA015704-30, and by grant RO1CA84069 to B.E.C.     

Comparative analysis of multilineage properties of mesenchymal stromal cells derived from fetal sources shows an advantage of mesenchymal stromal cells isolated from cord blood in chondrogenic differentiation potential

Background aimsCord blood (CB) and amniotic fluid (AF) could represent new and attractive mesenchymal stromal cell (MSC) sources, but their potential therapeutic applications are still limited by lack of standardized protocols for isolation and differentiation. In particular, chondrogenic differentiation has never been deeply investigated.MethodsMSCs were obtained from CB and AF samples collected during cesarean sections at term and compared for their biological and differentiation properties, with particular interest in cartilage differentiation, in which quantitative real-time polymerase chain reaction and immunohistochemical analyses were performed to evaluate the expression of type 2 collagen, type 10 collagen, SRY-box9 and aggrecan.ResultsWe were able to isolate MSCs from 12 of 30 (40%) and 5 of 20 (25%) CB and AF units, respectively. Fluorescence in situ hybridization analysis indicated the fetal origin of isolated MSC strains. Both populations expressed mesenchymal but not endothelial and hematopoietic markers, even though we observed a lower expression of human leukocyte antigen (HLA) I in CB-MSCs. No differences in proliferation rate and cell cycle analysis could be detected. After osteogenic induction, both populations showed matrix mineralization and typical marker expression. Under chondrogenic conditions, pellets derived from CB-MSCs, in contrast with AF-MSCs pellets, were significantly larger, showed cartilage-like morphology and resulted positive for chondrocyte-associated markers, such as type 2 collagen, type 10 collagen, SRY-box9 and aggrecan.ConclusionsOur results show that CB-MSCs and AF-MSCs collected at term differ from each other in their biological and differentiation properties. In particular, only CB-MSCs showed a clear chondrogenic potential and thus could represent an ideal candidate for cartilage-tissue engineering.     

Elongated cells derived from rat mammary cuboidal epithelial cell lines resemble cultured mesenchymal cells in their pattern of protein synthesis

Two-dimensional gel electrophoresis has been used to identify polypeptide patterns characteristic of rat mammary cuboidal epithelial cells or mesenchyme-derived cells. Elongated cells and cell lines derived from cloned cuboidal epithelial cells in culture possess a polypeptide pattern which resembles that of the cultured mesenchymal cells rather than that of the cuboidal epithelial cells from which they were derived. These elongated converts also resemble cultured mesenchymal cells in possessing a Triton-insoluble matrix in which vimentin and not prekeratin predominates.     

Dendritic cells reduce number and function of CD4+CD25+ cells in cytokine-induced killer cells derived from patients with pancreatic carcinoma

Aim and background: CD4⁺CD25⁺ cells are described as professional regulatory/suppressor T cells that are crucial for the prevention of spontaneous autoimmune diseases. They play an important role in maintaining a balanced peripheral immune system. On the other hand, it has been suggested that regulatory T cells (Treg) suppress antitumor immune responses after tumor-specific vaccinations. Therefore, we determined the percentage of regulatory T cells in cytokine-induced killer (CIK) cells, an effector cell population with high impact for adoptive immunotherapeutic strategies. Results: CIK cells showed strong induction of CD4⁺CD25⁺ cells with high secretion of interleukin 10 (IL-10) after unspecific stimulation of the TCR complex and stimulation with interleukin 2. Depletion of CD25⁺ cells led to an increase in cytotoxic activity and a reduction of IL-10 release. A more pronounced reversal of suppression could be induced by coculture of CIK cells with dendritic cells (DCs). After coculture of CIK cells with DCs, the number of CD4⁺CD25⁺ cells as well as the IL-10 concentration in the supernatant decreased, and the cytotoxic activity against pancreatic carcinoma cells increased. This was shown for cells from healthy donors as well as for cells from patients with pancreatic carcinoma. Conclusion: Our established effector cells possess some regulatory features induced by unspecific TCR-activation that could be prevented by coculture with DCs. CIK cells have desirable properties for immunotherapeutical approaches, especially after coculture with DCs, which could be used additionally for induction of a specific immune response.     

Progress in outgrowth culture from rabbit tracheal explants: Balance between proliferation and maintenance of differentiated state in epithelial cells

Summary Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively. This work was supported by DRET and by CIFRE grant awarded to S. R.     

Inhibition of lipopolysaccharide-induced release of interleukin-8 from intestinal epithelial cells by SMA, a novel inhibitor of sphingomyelinase and its therapeutic effect on dextran sulphate sodium-induced colitis in mice

Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The use of SMase inhibitors may offer new therapies for the treatment of the LPS- and cytokines-related inflammatory bowel disease (IBD). We synthesized a series of difluoromethylene analogues of SM (SMAs). Here, we show that LPS efficiently increases the release of IL-8 from HT-29 intestinal epithelial cells by activating both neutral SMase and nuclear factor (NF)-kappaB in the cells. The addition of SMA-7 suppressed neutral SMase-catalyzed ceramide production, NF-kappaB activation, and IL-8 release from HT-29 cells caused by LPS. The results suggest that activation of neutral SMase is an underlying mechanism of LPS-induced release of IL-8 from the intestinal epithelial cells. Ceramide production following LPS-induced SM hydrolysis may trigger the activation of NF-kappaB in nuclei. Oral administration of SMA-7 (60 mg/kg) to mice with 2% dextran sulfate sodium (DSS) in their drinking water, for 21 consecutive days, reduced significantly the severity of colonic injury. This finding suggests a central role for SMase/ceramide signaling in the pathology of DSS-induced colitis in mice. The therapeutic effect of SMA-7 observed in mice may involve the suppression of IL-8 production from intestinal epithelial cells by LPS or other inflammatory cytokines.     

Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen

Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo.     

Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme-dispersed male and female transgenic fetal rabbit gonads of 18–22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two-cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non-injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (α-antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co-transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8 to 16-cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines. © 1996 Wiley-Liss, Inc.