Phenotypic Plasticity of Retinal Pigment Epithelial Cells from Adult Human Eye <Emphasis Type="Italic">In Vitro</Emphasis>

Phenotypic plasticity of retinal pigment epithelial cells from adult human eye was studied by immunohistochemical methods under different culturing conditions. It was found that retinal pigment epithelium in adult human eye is a heterogeneous population of cells demonstrating different behavior in vitro. Some cells retain epithelial morphology for a long time in culture, while others are rapidly transformed into fibroblast-like cells and synthesize proteins typical of proneural, neural, glial, and photoreceptor cells. However, irrespective of initial morphological features differentiation of retinal pigment cells can be modulated by varying culturing conditions.     

Effect of Bioregulators Isolated from the Liver,Blood Serum,and Bile of Mammals on the State of Newt Liver Tissue in Organotypic Culture

We developed models of in vitro organotypic culturing of newt liver tissue with and without adhesion to the substrate. The effects of bioregulators isolated from mammalian liver, blood serum, and bile were studied on the developed models and their specifi city was demonstrated. The state of the liver was evaluated by the area of clusters of pigmented cells and by the number of mitoses in the connective tissue cells of the cortical layer. These bioregulators exhibited their biological effects only under conditions of roller organotypic culturing of newt liver tissue.     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbonstripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbonstripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that (1) the hormones extracted from bovine fetal calf serum by carbonstripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and (2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Studies in vitro on the mechanism of the epithelial/mesenchymal interaction in the early fetal thymus

The effect of the mesenchyme on early thymus development was investigated in vitro by culturing tissue recombinants of the epithelium and mesenchyme derived from the earliest fetal thymus primordium. The thymus mesenchyme promoted the development of the epithelium as assessed by expression of epithelial surface molecules such as major histocompatibility complex (MHC) class I and class II antigens; mesenchymes of other organs were similarly effective. We looked for the culture conditions in which the epithelium could normally express the MHC class II antigen without the mesenchyme and found that the insulin-like growth factors — I and — II were able to support epithelial development under serum-free conditions. When we cultured the prospective thymus region of a day 9 embryo which had not initiated lymphoid precursor migration, MHC class II antigen was expressed on the epithelium, indicating that lymphoid precursors are not required for early epithelial differentiation. This system provides a means to dissect the complex tissue interactions during the earliest stages of thymus development.     

Infection of stromal and hemopoietic precursor cells with lentivirus vector <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

We developed a method for gene transfer into mesenchymal stromal cells. Lentivirus vector containing green fluorescent protein gene for labeling stromal and hemopoietic precursor cells was obtained using two plasmid sets from different sources. The vector was injected into the femur of mice in vivo and added into culture medium for in vitro infection of the stromal sublayer of long-term bone marrow culture. From 25 to 80% hemopoietic stem cells forming colonies in the spleen were infected with lentivirus vector in vivo and in vitro. Fibroblast colony-forming cells from the femoral bones of mice injected with the lentivirus vector carried no marker gene. The marker gene was detected in differentiated descendants from mesenchymal stem cells (bone cavity cells from the focus of ectopic hemopoiesis formed after implantation of the femoral bone marrow cylinder infected with lentivirus vector under the renal capsule of syngeneic recipient). In in vitro experiments, the marker gene was detected in sublayers of long-term bone marrow cultures infected after preliminary 28-week culturing, when hemopoiesis was completely exhausted. The efficiency of infection of stromal precursor cells depended on the source of lentivirus. The possibility of transfering the target gene into hemopoietic precursor cells in vivo is demonstrated. Stromal precursor cells can incorporate the provirus in vivo and in vitro, but conditions and infection system for effective infection should be thoroughly selected. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 1, pp. 25–28, January, 2007     

A New Approach to Evaluation of Osteogenic Potential of Mesenchymal Stromal Cells

We developed a new approach to evaluation of the intensity of osteogenic differentiation of mesenchymal stromal stem cells based on measurement of optical density of mesenchymal stromal cell cultures in a concentration range 625–10,000 cells per well after their culturing in osteogenic induction medium and staining of calcium deposits by the method of von Kossa. The proposed method allows comparative semiquantitative evaluation of osteogenic properties of mesenchymal stem cells depending on tissue sources (bone marrow, adipose tissue, placenta), in vitro cell density, number of passages, duration of culturing, and concentration of serum growth factors in the microenvironment. The developed approach makes it possible to compare functional activity of mesenchymal stromal cells in various pathologies. The proposed method can be used in traumatology and orthopedics for improving the efficiency of transplantation of mesenchymal stromal cells for stimulation of reparative osteogenesis. Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 4, pp. 219–225, November, 2008     

Phenotypic Modification of Human Airway Epithelial Cells in Air–Liquid Interface Culture Induced by Exposure to the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen. We used an air–liquid interface epithelial cell culture system to model changes associated with NNK exposure relative to pathologies documented in human tobacco-related illnesses. Although in vitro systems exhibit certain limitations, they often offer accentuation of subtle pathologies. While the distribution of cell types in control cultures typically favors the ciliated cell phenotype, NNK-exposed cultures transitioned to non-ciliated cell phenotypes as well as reflecting features consistent with squamous metaplasia. We conclude that NNK impacts normal growth and differentiation of human airway epithelium in a short interval of time in vitro.     

Study of morphofunational peculiarities of fibroblasts isolated from human vertebral growth plates during <Emphasis Type="Italic">in vitro</Emphasis> culturing

We studied structural and functional peculiarities of growth zone chondroblasts isolated from human fetal spines on gestation week 12 and cultured in vitro over 4 passages. The morphology of chondroblasts of different differentiation degree was described. It was found that the population of chondroblasts had certain features determined by changes in the relative content of cells of different differentiation degree. The data suggest that culturing conditions affect cell differentiation and the possibility of using primary human chondroblast culture as the experimental model of differentiating human vertebral growth plate chondroblasts in vitro. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 3, pp. 154–158, July, 2007     

6th Pan Pacific Connective Tissue Societies Symposium Abstracts

Although lung epithelial cells directly attach to the basement membrane underneath in vivo, harvested epithelial cells are typically cultured on type I collagen gel (Col I-gel) in vitro. Recently we developed new culture substratum, designated as “synthesized Basement Membrane” (sBM), that has bared lamina densa on fibrillar collagen. To validate the usefulness of sBM substratum in airway tissue reconstitution in vitro, we cultured rat tracheal epithelial cells on sBM substratum and Col I-gel. When starting the air-liquid interface culture, most of the epithelial cells were squamous and positive for the basal cell marker cytokeratin 14 (CK14). After 14 days on sBM substratum, CK14-positive cells differentiated not only to Clara and mucous cells, but also to ciliated cells. Those differentiated cells formed pseudostratified-like epithelium and the remaining CK14-positive cells were polarized to the basal side. However, on Col I-gel, the CK14-positive cells were still squamous and not polarized, and ciliated cells did not appear. In conclusion, we established a new culture model on sBM substratum in which basal cells could differentiate to ciliated cells. The application of sBM substratum is useful in the study of the airway epithelial cell differentiation in vitro.     

Homocysteine toxicity in organotypic cultures of rat retina

Effects of homocysteine in toxic concentrations on retinal neurons were studied in vitro. In organotypic roller cultures of postnatal (8–12-day-old) and adult rat retina homocysteine caused multiple damage to neurons in the outer nuclear layer, in deep compartments of the inner nuclear layer, and ganglion cell layer. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 4, pp. 458–461, April, 2006     

Quantitative morphology of the central fovea in the primate retina

Electron micrograph composites of tangential sections of the fovea centralis of three cynomolgus monkeys (Macaco, irus) and one baboon (Papio anubis) were used to determine the spatial density of the principal retinal cells. In the center of the foveola, the density of cones ranged from 113,000 to 230,000/mm², and pigment epithelial cells from 4,900 to 7,000/mm². At a distance of 500μm from the foveolar center the density of the cone cell pedicles ranged from 29,000 to 36,300/mm², and the density of horizontal cells ranged from 19,000 to 25,100/mm². Densities of bipolar, Muller, and amacrine cells were determined in only two monkeys and in the baboon. The fact that the cone cell pedicles have a larger diameter than the foveolar cones explains the geometry of the fovea. The morphology of the junction between foveolar cone outer segments and the pigment epithelium reflects the complex metabolism of this functional unit. The comparison with the peripheral primate retina suggests that the densities of horizontal and bipolar cells, but not of amacrine and Muller cells, are correlated with the density of cone cell pedicles.     

An in vitro model for the study of human implantation

Citation Holmberg JC, Haddad S, Wünsche V, Yang Y, Aldo PB, Gnainsky Y, Granot I, Dekel N, Mor G. An In vitro model for the study of human implantation. Am J Reprod Immunol 2012; 67: 169–178 Problem Implantation remains the rate‐limiting step for the success of in vitro fertilization. Appropriate models to study the molecular aspects of human implantation are necessary in order to improve fertility. Methods First trimester trophoblast cells are differentiated into blastocyst‐like spheroids (BLS) by culturing them in low attachment plates. Immortalized human endometrial stromal cells and epithelial cells (ECC‐1) were stably transfected with GFP or tdTomato. Co‐culture experiments were monitored using Volocity imaging analysis system. Results This method demonstrates attachment and invasion of BLS, formed by trophoblast cells, into stromal cells, but not to uterine epithelial cells. Conclusion We have developed an in vitro model of uterine implantation. The manipulation of this system allows for dual color monitoring of the cells over time. Additionally, specific compounds can be added to the culture media to test how this may affect implantation and invasion. This model is a helpful tool in understanding the complexity of human implantation.     

Cytologic Observations on Trypsinized Cells Obtained from Human Endometrial Epithelium in Tissue Culture

Abstract

Although epithelial cells have been successfully grown from tissue culture of endometrial explants, they survived for only a few weeks before fibroblasts overgrew and eliminated the epithelium. We describe a method for growing endometrial epithelium from menstrual blood and passing them using 0.25% trypsin in EDTA; cells survived in vitro for 24 weeks. The morphology of the processed cells differed from that observed in vitro: trypsinized cells were rounded with long surface processes and microvilli; nuclei were irregular and contained a macronucleus; the cytoplasm appeared very granular. Immunocytochemistry revealed 90–95% positivity for anti-keratin; EM confirmed the light microscopic findings. Nuclei were large and irregular with a macronucleus and cytoplasmic organelles. The most striking feature of these cells was the abundance of rough endoplasmic reticulum, surface projections, and microvilli.

Endometrial epithelium undergoing trypsinization is not irreversibly damaged and retains its epithelial characteristics although it may be mechanically disrupted as a tissue and separated into single cells or small groups of cells. (The J Hislotechnol 10:233, 1987.)     

Intracellular replication of fusobacteria requires new actin filament formation of epithelial cells

We examined survival and replication of fusobacteria inside epithelial cells. Subconfluent cultures of HaCaT keratinocytes were infected with five bacterial strains representing three Fusobacterium species: F. nucleatum, F. necrophorum, and F. mortiferum. Adhesion and invasion of the bacteria were assayed before and after antibiotic treatment that killed the adhered and extracellular bacteria. The number of live fusobacteria was examined by bacterial culturing after sonication of the epithelial cells. The role of host cell cytoskeleton functions was examined by treating the epithelial cells with cell function inhibitors. Number of viable epithelial cells was measured with the CellTiter96 kit. The tested Fusobacterium species adhered to and invaded the epithelial cells, and multiplied intracellularly for several hours. Thereafter, the intracellular number of bacteria rapidly declined. Concomitantly, viable fusobacteria were detected in the culture medium. Treatment of the infected epithelial cells with an actin formation inhibitor markedly reduced the number of living intracellular fusobacteria. Newly formed actin filaments were seen by confocal microscopy in the epithelial cells associated with the invaded bacteria. Fusobacteria infection did not reduce the number of viable epithelial cells in culture. Thus, fusobacteria are able to adhere to and invade epithelial cells, and survive under aerobic conditions. This property may enable them to survive in mucosa and participate in various disease processes of oral and pharyngeal tissues.     

Experimental Models for Culturing of Eye Tissues of <Emphasis Type="Italic">Pleurodeles Waltl</Emphasis> for Evaluation of Specific Effects of Sclera Bioregulator in Ultra-Low Doses

Experiments on stationary culture of posterior eye and roll-bottle culture of the whole eye from adult water lizards Pleurodeles waltl showed that sclera bioregulator produces a stabilizing effects on adhesion interactions between the sclera, choroid, and pigment epithelium and on the maintenance of viability of sclera fi broblasts and pigment epithelium cells.     

Short-term biological safety of a photoelectric dye used as a component of retinal prostheses

We have designed a new type of retinal prosthesis with a photoelectric dye that transfers photon energy to generate electric potentials. The purpose of this study was to test the safety of a photoelectric dye, 2-[2-[4-(dibutylami no)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide (NK-5962), used for retinal prostheses. The retinal cells, derived from chick neurosensory retinas at the 12-day embryonic stage, were a mixed population of retinal neurons and glial cells, and were cultured for 2 days either under protection from light or under continuous light exposure at 230 lux for 9 h daily in the presence of the photoelectric dye at varying concentrations (1.6 × 10⁻⁵, 1.6 × 10⁻⁶, and 1.6 × 10⁻⁷ M) to assess cell viability by staining live cells and dead cells. Dispersed retinal pigment epithelial cells at the same embryonic stage were incubated with the photoelectric dye at varying concentrations (6.6 × 10⁻⁵, 6.6 × 10⁻⁶, and 6.6 × 10⁻⁷ M) for 4 h under protection from light or under continuous light exposure at 320 lux to assess cytotoxicity by measuring the activity of lactate dehydrogenase leaking from cells. The majority of retinal cells were alive with only a small percentage of dead cells under the dark condition or the light condition in the presence or the absence of the photoelectric dye. The percentage of dead cells was significantly smaller at higher concentrations of the photoelectric dye (P = 0.0183, two-factor analysis of variance), while the percentage of dead cells was not significantly different between the dark condition and the light condition (P = 0.3102). Percent cytotoxicity values were negative, indicating protective effects in all groups of retinal pigment epithelial cells incubated with varying concentrations of the photoelectric dye. The photoelectric dye showed no cytotoxicity to chick retinal cells or retinal pigment epithelial cells on short-term exposure. In addition, this photoelectric dye might have protective effects on both types of cells.     

Roller Organotypic Cultures of Postnatal Rat Retina

Floating retinal sections from 7-12-day-old rats form ball-shaped retinal bodies during roller culturing. Histological studies of serial sections of retinal bodies showed that their outer surface is formed by the retina completely retaining organotypic cytoarchitectonics. Some retinal bodies have laminar structure consisting of several layers of the retina. At the initial stages of culturing some retinal bodies contain a cavity, which later is completely obliterated due to the growth of axons of ganglion cells and migration of glial cells and fibroblasts. This study demonstrated the possibility of long-term survival, differentiation, and in vitro axonal regeneration of ganglion cells, the main retinal efferent neurons, which can provide the basis for investigation of pathology and drug correction of injuries and stimulation of regeneration of these cells in experimental glaucoma models.     

Pathology of the Pigment Epithelium and Retina in Rabbits Poisoned with Lead

Multifocal lesions of the retinal pigment epithelium were observed in rabbits fed a diet contaning 0.5% lead subacetate for periods of up to 2 years. Groups of pigment epithelial cells became congested with a lipofuscin pigment which was apparently derived from phagosomes of rod outer segments. Lipofuscin granules displaced melanin granules from the apical surface of the retinal pigment epithelial cells and resulted in conspicuous brown pigmentation of these cells in albino animals. Migration of macrophages and pigment epithelial cells into the subretinal space was common in affected areas. This pathology was not observed in the pigment epithelium of the ora serrata, or ciliary body. At the latest time periods, the abnormal lipofuscin pigmentation subsided, and degeneration of photoreceptors occurred. The pathogenesis of the lesions is discussed.     

Alternative Method for Primary Nasal Epithelial Cell Culture Using Intranasal Brushing and Feasibility for the Study of Epithelial Functions in Allergic Rhinitis

Purpose

Although differentiated normal human nasal epithelial (NHNE) cells can be used to study the role of human nasal epithelium, there is a need for effective culture models of nasal epithelium in sinonasal disease status, including allergic rhinitis (AR). We aimed to examine the feasibility of intranasal brushing for culture of nasal epithelial cells in AR patients and to verify the hypothesis that allergic nasal epithelial (ARNE) cells differ in histologic and physiologic characteristics.

Methods

We established a system for isolating (via intranasal brushing) and culturing (with air-liquid interface, ALI) nasal epithelial cells from healthy volunteers (n=8) and AR patients (n=8). We used this system to compare the histologic findings and physiologic characteristics of NHNE and ARNE.

Results

The histology results showed that fully differentiated ALI culture was obtained at least 14 days after confluence and that both ciliated and secretory cells were well differentiated in ALI culture using nasal brushing. The histology results of ARNE culture were significantly different from NHNE. The number of ciliated cells was lower, and secretory cells were more dominant in ARNE cell culture compared to NHNE cells. We also observed, by electron microscopy, loose tight junctions and short cilia in cultured ARNE cells. In addition, the mRNA level of TSLP which was one of the epithelial-derived allergic cytokines was significantly higher, and the expressions of genes involved in ciliogenesis were lower in cultured ARNE cells without allergen stimulation.

Conclusions

Our findings suggest that ALI culture of ARNE cells using intranasal brushing may be an alternative method for epithelial cell culture in AR patients and that cultured ARNE cells will be useful for in vitro studies of the mechanisms at play during AR because they maintain unique allergic characteristics.