COMPARISON OF NEURONAL AND LENS PHENOTYPE EXPRESSION IN THE TRANSDIFFERENTIATING CULTURES OF NUERAL RETINA WITH DIFFERENT CULTURE MEDIA*

The effects of three different culture media (Eagle's MEM, F-12 and L-15) on the transdifferentiation of 8-day chick embryonic neural retina into lens cells, were examined with respect to the expression of two phenotypes. One type referred to neuronal specificity (as represented by the level of cholineacetyl-transferase, CAT, activity) and the other to lens specificity (as represented by content of α-and δ-crystallin). In 7-day cell cultures before the visible differentiation of lentoid bodies, CAT activity was detected in all media. But, its level was about 9 times higher in cultures with L-15 than in those with MEM and 3 times higher than in F-12. In 26-day cultures, CAT activity was practically undetectable. The production of α-and δ-crystallin was detected in cultures at 26 days. There were quantitative differences in the crystallin content with different media, and it was highest in cultures with L-15. The results indicate that conditions most favourable to the maintenance of the neuronal specificity in cell cultures of neural retina, can also support the most extensive transdifferentiation. The possibility of direct transdifferentiation of once neuronally specified cells into lens cells in cultures with L-15 has been suggested to explain the present results.     

Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture.

The possible multipotential nature of the neural retina of early chick embryos was examined by the technique of clonal cell culture. Cultures were prepared from cells dissociated from freshly excised neural retinas of 3.5-day-old chick embryos or from cells harvested from primary highdensity cultures. The following four colony types were obtained: colonies differentiating into “lentoid bodies”; colonies with pigment cells; colonies with both “lentoid bodies” and pigment cells; and colonies comprised entirely of unidentifiable cells. Neuronal differentiation occurred frequently in the early stages of culture (up to about 10 days). In some of these neuronal colonies, “lentoid bodies” and, rarely, both “lentoid bodies” and pigment cells differentiated after a further culture period of up to 30 days. Secondary colonies established from primary colonies after 9–10 days demonstrated that these original colonies fell into four different categories: those giving rise to secondary colonies containing only “lentoid bodies,” those giving rise to pigmented colonies only, those developing both lentoid and pigmented colonies, and finally those which gave rise to secondary colonies of all three types, lentoid, pigmented, and mixed colonies. When primary pigmented colonies were recloned at about 30 days after inoculation, the differentiated pigment cells transdifferentiated into lens. Whether multispecific colonies were really of clonal origin or not is discussed. The possible presence of a multipotent progenitor cell able to give rise to multispecific clones in the neural retina of 3.5-day-old chick embryos is suggested. A sequence of differentiation starting from multipotent neural retinal cells to be terminated with lens through the differentiation of neuronal and pigment cells is hypothetically proposed.     

EFFECTS OF CULTURE MEDIA ON THE "FOREIGN" DIFFERENTIATION OF LENS AND PIGMENT CELLS FROM NEURAL RETINA IN VITRO

Neural retinal cells of 3.5-day-old quail embryos were cultured as a monolayer to examine their potentials for differentiation in vitro. The "foreign" differentiation into lentoid and pigment cells was much affected by the choice of medium (Eagle's MEM and Ham's F–12); in Eagle's MEM, neural retinal cells differentiated extensively into lentoid bodies and pigment cells, as previously reported in cultures of chick neural retinal cells, while in Ham's F–12, though the cells proliferated as well as in Eagle's MEM, the "foreign" differentiation is inhibited. When primary cultures were transferred to secondary cultures, the occurrence of "foreign" differentiation did not depend on the medium used for the primary culturing, but wholly on the medium used for secondary cultures. This difference in differentiation in two different media was quantitatively substantiated by measuring the amounts of α-, δ-crystallins and melanins of cultured cells.     

Can Once Neuronally Differentiated Cells of Neural Retina Be Lentoidogenic in Cell Culture?*

Cells dissociated from neural retina of 3.5-day-old chick embryos transdifferentiated extensively into lens cells under the conditions of a cell culture for 3 to 4 weeks. In early satges of cell culture by about 10 days, cultures consisted of small round cells often with cytoplasmic processes(N-cells) and flattened epithelial cells (E-cells). Only N-cells were stained with a fluorescent dye Merocyanine 540. When cells harvested from early cultures were separated into two fractions by centrifugation in Percoll gradient, the specific activity of choline acetyltransferase was much higher in the fraction consisting mainly of N-cells than in other fraction mainly of E-cells. Continuous daily observations as well as cinematographic observations of living cultures indicate that lentoid bodies were often formed in the locations where clusters of N-cells had been found in early stages of culturing. The possibility of transdifferentiation of N-cell clusters into lentoid bodies is discussed.     

Biochemical and immunological studies of lentoid formation in cultures of embryonic chick neural retina and day-old chick lens epithelium

During long-term cell culture of 8-day embryonic chick neural retina, lentoid bodies containing lens crystallins are developed. Although very low levels of crystallin can be detected in the embryonic neural retina, gross synthesis of each major crystallin class (α, anodal β, cathodal β, and δ) begins only after 12–16 days in culture. This occurs at least 10 days before lentoid bodies can be distinguished by eye. The concentration of each crystallin class was determined during lentoid development in cultures of both neural retina and lens epithelium. The proportions of crystallins in lentoid-containing cultures do not resemble those of embryonic lens fibres. Comparisons between two chick strains (N and Hy-1) differing in their growth rates revealed several differences in the crystallin compositions of lentoid bodies. These differences imply independent quantitative regulation for most or all of the crystallins.     

Differential effects of culture media on normal and foreign differentiation pathways followed by chick embryo neuroretinal cells in vitro

Three different culture media, Ham's F-12, medium 199, and Eagle's minimal essential medium (MEM), were compared with respect to the expression of neuronal (choline acetyl transferase activity: CAT) and glial (hydrocortisone-induced glutamine synthetase activity; GSase) markers of normal differentiation in cultures of 9-day chick embryo neuroretinal cells, and also with respect to the accumulation of a lens marker (delta crystallin) during so-called 'transdifferentiation' in these cultures. MEM allows transient expression of both CAT and GSase activities in early cultures, but also permits extensive delta crystallin accumulation at later stages. F-12 medium gives somewhat higher levels of CAT and GSase activities, the former being noticeably prolonged as compared with parallel MEM cultures; delta crystallin accumulation, however, is largely inhibited in F-12 cultures. By contrast, medium 199 permits only low levels of CAT and GSase activities, perhaps because the neuronal cells are distributed individually over the glial cell sheet in 199 cultures, rather than forming aggregates as in MEM or F-12 cultures. Medium 199 also blocks delta crystallin accumulation. The results of medium changeover between 'transdifferentiation'-permissive (MEM) and non-permissive (199, F-12) conditions suggest: (a) that potential lens precursor cells (whatever their nature) survive in F-12 medium for prolonged periods without extensive expression of the lens phenotype; (b) that such precursor cells become committed to subsequent differentiation as lens cells between 10 and 20 days of culture in permissive MEM medium (as judged by the accumulation of delta crystallin following transfer into F-12); and (c) that medium 199 can block expression of the lens phenotype even in cells already committed (by the above criteria) to lens differentiation, as for instance after 30 days of preculture in MEM.     

Differential effects of culture media on normal and foreign differentiation pathways followed by chick embryo neuroretinal cells in vitro

Abstract. Three different culture media, Ham's F-12, medium 199, and Eagle's minimal essential medium (MEM), were compared with respect to the expression of neuronal (choline acetyl transferase activity: CAT) and glial (hydrocortisone-induced glutamine synthetase activity; GSase) markers of normal differentiation in cultures of 9-day chick embryo neuroretinal cells, and also with respect to the accumulation of a lens marker (δ crystallin) during so-called 'transdifferentiation' in these cultures.
MEM allows transient expression of both CAT and GSase activities in early cultures, but also permits extensive δ crystallin accumulation at later stages. F-12 medium gives somewhat higher levels of CAT and GSase activities, the former being noticeably prolonged as compared with parallel MEM cultures; δ crystallin accumulation, however, is largely inhibited in F-12 cultures. By contrast, medium 199 permits only low levels of CAT and GSase activities, perhaps because the neuronal cells are distributed individually over the glial cell sheet in 199 cultures, rather than forming aggregates as in MEM or F–12 cultures. Medium 199 also blocks δ crystallin accumulation.
The results of medium changeover between 'transdifferentiation'-permissive (MEM) and non-permissive (199, F-12) conditions suggest: (a) that potential lens precursor cells (whatever their nature) survive in F-12 medium for prolonged periods without extensive expression of the lens phenotype; (b) that such precursor cells become committed to subsequent differentiation as lens cells between 10 and 20 days of culture in permissive MEM medium (as judged by the accumulation of δ crystallin following transfer into F-12); and (c) that medium 199 can block expression of the lens phenotype even in cells already committed (by the above criteria) to lens differentiation, as for instance after 30 days of preculture in MEM.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

pp60c-src expression in transdifferentiating cultures of embryonic chick neural retina cells

Chick embryo neural retinal cells transdifferentiate extensively into lens cells when cultured in Eagle's MEM containing horse and fetal calf sera (FHMEM). Such cultures express elevated levels of pp60c-src-associated tyrosine kinase activity relative to parallel cultures prevented from transdifferentiating by the addition of supplementary glucose (FHGMEM) or replacement of MEM by medium 199 (F199). Northern blotting and in vitro translation studies suggest that c-src mRNA levels are only slightly higher in late transdifferentiating (FHMEM) cultures as compared to parallel blocked (FHGMEM or F199) cultures. By immunocytochemical staining, we show that pp60c-src protein is largely localized in cell groups undergoing conversion into lens (i.e. expressing delta crystallin) in late FHMEM cultures. Initial studies of pp60c-src in chick lens tissues during development indicate that higher kinase activity is found in the epithelial cells relative to mature lens fibres. Thus pp60c-src may be expressed both during the differentiation of lens cells in vivo and during the transdifferentiation of neural retina cells into lens in vitro.     

Differentiation of lens and pigment cells in cultures of neural retinal cells of early chick embryos

Dissociated cells of neural retinas of 3.5-day-old chick embryos (stages 20–21) were cultured as a monolayer in order to examine their differentiation in vitro. These cells started to grow actively soon after inoculation and formed a confluent sheet within which neuroblast-like cells with long cytoplasmic processes were differentiated by 8 days. At about 16 days the differentiation of both lentoid bodies and foci of pigment cells was observed, while neuronal structure disappeared. The numbers of lentoid bodies and foci of pigmented cells continued to increase up to 30 days, when primary cultures were terminated. The increase in δ-crystallin content, as measured by quantitative immunoelectrophoresis assay using rabbit antiserum against δ-crystallin, was consistent with the increase in the number of lentoid bodies in cultures. The amount of α-crystallin per culture, estimated by the same technique as above, reached a maximum at 16 days and decreased slightly during further culture. The differentiation of both lentoid bodies and pigment cells was observed also in cultures of the second generation. The results demonstrate that cells of the undifferentiated neuroepithelium of 3.5-day-old embryonic retinas can achieve at least three differentiations, neuronal, lens, and pigment cells, in vitro. We discuss several differences between the present results and the previous ones from in vitro cultures of 8- to 9-day-old embryonic neural retinas.     

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ .     

Differentiation of lens in cultures of neural retinal cells of chick embryos

When dissociated cells of neural retinae of 8-day-old chick embryos were cultured, monolayer sheets of epithelial cells were obtained. These cells proliferated actively. After about 30 days of culture, both lentoid bodies and pigment cells were differentiated in all plates. In the second and the third generation cultures, both differentiations were also observed. Lentoid bodies showed positive immunofluorescence for fluorescein-isothiocyanate-conjugated antiserum against δ-crystallin. Molecular constituents of lentoid bodies were very similar to those of lenses developing in situ, as revealed by immunodiffusion tests. Several lines of evidence for the “neural retinal” origin of lentoid bodies, as opposed to their being derived from lens cells inadvertently included in the original culture inocula are given. Some implications of the present results for the problem of “determination” are discussed.     

Microencephaly and microphthalmia in rat fetuses by busulfan

Microencephaly and microphthalmia in the embryos/fetuses from rats exposed to busulfan were histopathologically examined. Busulfan was intraperitoneally administered at 10 mg/kg on gestation days (Days) 12, 13 and 14, and then embryos/fetuses were harvested on Days 14.5, 15, 16 and 21. In the treated group on Day 21, all fetuses were small with reduced body weight, with microencephaly and microphthalmia. On Days 14.5, 15 and 16, apoptotic cells were increased in the neuroepithelium and the neural retina with a width reduction and a decrease in cell density, and the lens epithelial cells histopathologically. Mitotic inhibition was observed in the neuroepithelium, neural retina and equatorial zone of the lens. On Day 21, the cerebral cortex and the retina became markedly thinner. The lens fibers showed swollen, fragmentary and vacuolar formation in the cranial portion accompanied with small lens sizes. The anti-proliferative effects of busulfan brings about a lack of cell populations required for the normal organogenesis of the brain and eye, and leads to microencephaly and microphthalmia, featuring hypoplasia of cerebrum and hypoplasia of retina and lens with cataract, respectively.     

Differentiation of Striated Muscle Fibers in Monolayer Cultures of Rat Anterior Pituitary

Striated muscle fibers appeared in monolayer cultures of rat anterior pituitary cells maintained in αMinimum Essential Medium (αMEM). As muscle differentiation in cultures of pituitary cells under ordinary conditions has not hitherto been reported, an in vitro study was undertaken to determine what factor(s) is responsible for this myogenesis. When dispersed anterior pituitary cells were culrured in three different media, αMEM, Medium 199 and Dulbecco's Modified Eagle Medium (DMEM), only αMEM induced a high incidence of striated muscles. The nature of the serum (fetal calf, calf and horse) and its concentration (1–10%) did not affect myogenesis.
In monolayers in αMEM, the sequence of differentiation of striated muscle was as follows: 1) Elongated cells, resembling myoblasts appeared; 2) these cells fused; and finally 3) cross striations appeared. Rhythmic contraction was most intense in striated muscle fibers, but it was also obsrved in myotubes without cross striations and even in myogenic cells before fusion. The possible origin of muscles in these pituitary cultures is discussed.     

Retinal neural progenitors express topographic map markers

Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Retinal ganglion cell axons project to visual centers of the brain forming circuits in precise topographic order. In chick, dorsal retinal neurons project to ventral optic tectum, ventral neurons to dorsal tectum, anterior neurons to posterior tectum and posterior neurons to anterior tectum; forming a continuous point-to-point map of retinal cell position in the tectal projection. We found that when stem cells derived from ventral retina were implanted in dorsal host retina, the stem cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors exhibit topographic markers of cell position in retina. Indeed, retinal neural progenitors express topographic markers: dorsal stem cells expressed more Ephrin B2 than ventral stem cells and, conversely, ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells. The fact that neural progenitors express topographic markers has pertinent implications in using neural stem cells in cell replacement therapy for replacing projecting neurons that express topographic order, e.g., analogous neurons of the visual, auditory, somatosensory and motor systems.     

THE DIFFERENTIATING ABILITY OF RAT LENS EPITHELIAL CELLS IN CELL CULTURE

Dissociated cells of lens epithelia of adult rats were monolayerly cultured in vitro. After about 15–20 days' period of active cell growth, such characteristic structures that correspond to "lentoid bodies" described previously in chick cultures were formed. These structures consisted of elongated cells, ultrastructural profile of which was similar with lens fiber. The presence of gamma-crystallin, a marker molecule specific to mature lens fiber, was confirmed in these elongated cells by means of fluorescent antibody technique. The differentiation of lens fiber in vitro was also recognized in clones originating from single lens epithelial cells cultured at very low cell density.     

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.     

Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma

From a transplantable mouse teratoma it has been possible to derive an established keratinizing cell line (XB) which grows well in cultures containing lethally irradiated 3T3 fibroblasts at the correct density. Single cells of the keratinizing line grow into colonies each consisting of a stratified squamous epithelium. The keratinizing nature of the colonies has been demonstrated by specific staining with Rhodanile blue, and by light and electron microscopy of sections through the colonies. A function of fibroblasts appears to be a strict requirement for keratinization and an important though less strict requirement for cell growth. The fibroblast function can be carried out by medium harvested from 3T3 cultures.It is possible to detect keratinizing colonies in primary cultures of disaggregated teratoma cells combined with 3T3 cells. Such colonies appeared in cultures of a transplantable teratoma with an overall frequency of 6 × 10⁻⁶ of the cells plated. Nonkeratinizing colonies of cells with otherwise very similar appearance were about 10 fold more abundant. Since both the keratinizing and the related nonkeratinizing colonies can be identified in the living state, it is possible to isolate them from the primary cultures.     

A Modified In vitro Method to Obtain Pure Astrocyte Cultures Induced from Mouse Hippocampal Neural Stem Cells Using Clonal Expansion

The aim of the present study was to produce astrocyte cultures of high purity from mouse hippocampal neural stem cells and to compare their in vitro properties with those isolated from enriched mixed glial cultures prepared from mouse hippocampus, which are commonly contaminated by microglia. We produced primary cultures of newborn mouse hippocampal neural stem cells, which have the potential to differentiate into astrocytes, neurons, and oligodendrocytes. We produced monoclonal neural stem cell colonies by limiting dilution. We induced astrocyte differentiation by plating the colonies on poly-l-lysine and culturing them in induction medium consisting of minimum essential medium/F12 supplemented with 10% fetal bovine serum and 100 ng/ml ciliary neurotrophic factor. We then further purified the cells by differential adherence and shaking at a constant temperature, followed by a second round of limiting dilution. Immunocytochemistry for glial fibrillary acidic protein showed that our method yielded 99.4 ± 0.5% pure astrocytes, whereas traditionally enriched mixed glial cultures yielded 94.2 ± 2% pure astrocytes. Induced cells resembled primary astrocyte cultures in functional properties such as cell proliferation rates and lack of tumorigenicity and p53, and expression of epidermal growth factor receptor, bystin, and nitric oxygen synthase. Our novel method of culture and purification of neural stem cells can therefore be used routinely for the primary culture of highly purified astrocytes from mouse hippocampus.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.