AN ANALYSIS OF DIFFERENTIATIVE CAPACITY OF PIGMENTED EPITHELIAL CELLS OF ADULT NEWT IRIS IN CLONAL CELL CULTURE

Singly dissociated cells from dorsal and ventral iris epithelia ( iris iridica ) of adult newts were cultured separately at clonal density to analyse their growth and differentiative capacity. Usually some attached cells began to proliferate on 12th day of culture, and grew with loss of melanosomes to form clonal cell colonies. Up to 30 days after inoculation, most of the clonal colonies formed typical epithelial monolayer sheets which consisted mostly of nonpigmented cells. Then, in some of those colonies, cells piled up together and form typical lens structures containing lens antigens. A month and a half after culturing, 30 to 40% of single iris cells, which had been previously marked, grew to form clonal colonies consisting of more than 100 cells. About 30% of these colonies expressed lens specificity and no significant differences in efficiency of colony formation and differentiation were detected between the dorsal cells and the ventral, suggesting that potent cells capable of transdifferentiating into lens cells are evenly distributed in all parts of the newt iris epithelium.     

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.     

A DEMONSTRATION OF LENS FORMING-CELLS IN NEURAL RETINA IN CLONAL CELL CULTURE

Clonal cultures with 1,000–3,000 cells were prepared from cells harvested from high density cultures of neural retina of 8-day-old chick embryos. About 1.14% and 0.31% of inoculated cells developed into recogniziable colonies in Eagle's MEM and in Ham's F-12 supplemented with fetal calf serum respectively. Of these colonies, lentoid bodies of authentic lens nature were differentiated in 10% and 33.52% in MEM and F-12 respectively. Cells harvested from high density cultures of the anterior and posterior portions of the neural retina were clonally cultured. Plating efficiency was much higher in the anterior cells than in the posterior ones and clonies with lentoid differentiation were developed only in clonal cultures of the anterior cells.     

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.     

The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikha?lov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.     

Transformation of Iris into Lens in vitro and its Dependency on Neural Retina

Upon lentectomy of adult newt eyes, the dorsal iris epithelium produces a cell population that develops into a new lens. The tissue transformation can be completed not only in the isolated lentectomized eye cultured as a whole, but also in the isolated newt normal dorsal iris combined with the retina of frog larvae in vitro. In this study, 93% of such cultures produced lens tissue made up of newt cells. Well-differentiated lens fibre cells were formed which showed positive immunofluorescence for gamma crystallins. When the isolated dorsal iris epithelium was cultured under the same conditions, well-differentiated lens tissue was again formed in 95% of the cases, suggesting that iris epithelial cells and not iris stromal cells are responsible for lens formation. In contrast, the combination of newt ventral iris with frog retina did not produce any newt lens tissue. No lens tissue was produced when the dorsal iris was cultured with newt spleen or lung, although a considerable number of iris epithelial cells became depigmented. Isolated normal dorsal iris or normal dorsal iris epithelium cultured alone infrequently produced a population of depigmented cells but failed to form lens tissue. On the basis of the present and earlier data, it is concluded that in Wolffian lens regeneration in situ , interaction of the iris epithelial cells with the retina plays a decisive role. However, it is suggested that the iris epithelial cells may have an inherent tendency towards lens formation, and that the factor(s) from the retina facilitates the realization of this tendency, rather than instructing the cells to produce lens. The reported experiments provide the first direct evidence for the existence of cellular metaplasia by demonstrating transformation of fully differentiated iris epithelial cells into lens cells.     

Direct evidence for transformation of differentiated iris epithelial cells into lens cells

Although it is generally assumed that the lens regenerated in the newt eye after complete lentectomy is formed by cells derived from the dorsal iris epithelium, experimental evidence so far obtained for this transformation does not rule out participation of cells from the dorsal iris stroma. When the normal dorsal iris epithelium of adult Notophthalmus (Triturus) viridescens was isolated and cultured in the presence of frog retinal complex, newt lens tissue was produced in 88% of cultures. These lens tissues were positive for immunofluorescence for lens-fiber-specific gamma crystallins as well as for total lens protein. On the basis of a study of stromal cells contaminating the samples of dorsal iris epithelium and a test for the lens-forming capacity in vitro of the dorsal iris stroma in the presence of frog retinal complex, it is concluded that lens formation observed in the above experiment is not dependent on the contaminating stromal cells. This implies that, in Wolffian lens regeneration, fully differentiated adult cells completely withdrawn from the cell cycle are transformed into another cell type. An additional culture experiment demonstrated that lens-forming capacity is not restricted to the dorsal half of the iris epithelium, but extends into its ventral half.     

In vitro analysis of cellular metaplasia from pigmented epithelial cells to lens phenotypes: a unique model system for studying cellular and molecular mechanisms of "transdifferentiation"

Pigmented epithelial cells (PECs) were dissociated from eyes of 8- to 9-day-old chick embryos and were cultured in EdF medium (Eagle's MEM supplemented with dialyzed fetal bovine serum) containing phenylthiourea (PTU) and testicular hyaluronidase (HUase). The PECs rapidly lost melanosomes as they proliferated and dedifferentiated in culture. These dedifferentiated PECs (dePECs) which did not manifest any identifiable specificity could be directed to one of two different differentiated phenotypes; viz., lens or pigment cells, depending upon subsequent culture conditions. Almost all dePECs began to synthesize melanin and redifferentiated to PECs by Day 10 of culture with EdF medium containing ascorbic acid (AsA). In contrast, the sister population of dePECs, when cultured at extremely high cell density with EdF medium containing PTU, HUase and AsA, synthesized delta-crystallin which is specific for lens. This transdifferentiation into lens cells occurred by Day 15 of culture. Using this culture system we are able to produce a homogeneous cell population with the potential for synchronous differentiation into either lens or pigment cell phenotype. The system is useful for studying mechanisms involved in cellular metaplasia.     

Developmental and physiological pattern of aldose reductase mRNA expression in lens and retina

Aldose reductase (AR), an enzyme which converts glucose to sorbitol, has been implicated in the pathogenesis of diabetic cataracts and retinopathy. The normal physiological role of this enzyme in ocular tissue, however, remains unclear. In a developmental study in the rat using in situ and Northern hybridization analyses, we have found that there is a high level of AR mRNA expression in optic cup and lens as early as embryonic day 13. Serial sections through whole embryos at this stage showed that the eye was the only site of AR mRNA hybridization. Levels of AR mRNA declined in the retina as differentiation proceeded and were very sparse there postnatally. As lens development progressed, epithelial AR mRNA levels remained high, especially in the germinative zone, which is the source of the cells that will become lens fibers, and in the bow region, where these cells undergo a dramatic morphogenetic differentiation into lens fibers. AR mRNA was undetectable in terminally differentiated lens fibers. Since it has been suggested that AR-catalyzed sorbitol production could be an osmoprotective device of lens epithelium during systemic hyperosmolar stress, AR mRNA levels from dehydrated hyperosmolar rats were compared with euvolemic control values, and no difference was found. In summary, AR appears to be of particular importance in the development of the eye, with its retinal role receding relative to lens as differentiation is completed. A continued high level of expression in lens epithelium in adulthood may be explained by the fact that lens tissue, unlike retina, normally continues to proliferate and differentiate after birth. The temporal and spatial pattern of distribution of AR mRNA is strongly suggestive of a role for this enzyme in lens fiber morphogenesis.     

Anchorage-independent growth of normal calf lens epithelial cells and effect of SV40 transformation on their growth properties

Calf lens epithelial cells cultured in vitro show growth properties usually associated with virally transformed fibroblasts. The lens cells are anchorage independent, forming colonies in agar, and show a low requirement for added mitogens. In dense culture they form multilayers and maintain a constant cell number by proliferation and shedding. Strains of lens cells transformed by SV40 virus have been obtained that show similar growth properties to the normal lens epithelium. The major effect of SV40 transformation is to increase the growth rate, final cell density and in vitro life-span of the lens cells and to inhibit the increase in size that occurs after 2-3 weeks of culturing the untransformed cells.     

GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye^1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment⁴; however the cells are still capable of forming the different cell types found within the neural retina^1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury⁵, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent^6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation^9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.     

Clonal analysis of vertebrate myogenesis: IV. Medium-dependent classification of colony-forming cells

The cell lineage of chick leg muscle between 3 and 12 days of development has been studied by use of an in vitro clonal assay. The assay permits distinctions to be made among various types of muscle-colony-forming cells (MCF cells) on the basis of their medium requirements and clonal morphology. Results suggest the sequential occurrence of at least four types of MCF cells, three of which require conditioned medium for their differentiation and one of which can form differentiated colonies in fresh medium.The nature of the “conditioned medium effect” was further investigated by the use of medium-switch experiments. By this process it was shown that the same populations of colony-forming cells attach and grow in fresh and conditioned medium and that the differentiation of colonies derived from conditioned-medium-requiring myoblasts is permitted by brief exposure to conditioned medium followed by culture in fresh medium. Further investigation indicated that during brief exposure to conditioned medium the gelatin-coated petri plate surface is altered such that differentiation of conditioned-medium-requiring colonies is allowed. We conclude that the conditioned medium effect involves a surface-mediated interaction between myoblasts and one or more conditioned medium components.     

The growth and morphological characteristics of undifferentiated and differentiated cells of the F9 mouse teratocarcinoma line

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.     

Control of lens epithelial cell survival

We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell- cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture.     

In VitroCulture System for Iris-Pigmented Epithelial Cells for Molecular Analysis of Transdifferentiation

Dissociated cells of the iris-pigmented epithelium (IPE) from a 1-day-old chick grew in monolayer culture and stably maintained their differentiated state when cultured with standard culture medium. After replacement of the control medium by EdFPH medium, which is effective in inducing dedifferentiation of retinal pigmented epithelium (RPE) cells, all cells rapidly lost pigment granules, proliferated intensively, and dedifferentiated. By further addition of ascorbic acid, dedifferentiated cells accumulated and formed a large number of lentoids. This system provides a useful opportunity for analyzing cellular and molecular mechanism involved in each step of transdifferentiation. Furthermore, Northern blot data indicates that the up-regulation of pax-6 gene could be an important event during lens regeneration as well as during normal lens development.     

Decreased turnover of phosphatidylinositol accompanies in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers

The in vivo differentiation of embryonic chicken lens epithelial cells into lens fibers is accompanied by a marked decrease in the rate of degradation of phosphatidylinositol. The present experiments were undertaken to determine whether a similar change in phosphatidylinositol metabolism occurs during in vitro lens fiber formation in cultured explants of embryonic chicken lens epithelia. Lens epithelial cells in the explants differentiate into lens fibers following the addition of fetal calf serum, insulin or chicken vitreous humor to the culture medium. The results show that phosphatidylinositol is degraded with a half-life of 3-6 h in cultured lens epithelia that are not stimulated to differentiate. In contrast, no degradation occurs for at least 6 h in lens epithelia stimulated to form lens fibers. The stabilization of phosphatidylinositol is apparent within 4 h after the onset of fiber cell formation, and thus represents an early event in differentiation. The rapid degradation of phosphatidylinositol in lens epithelia is accompanied by comparably rapid synthesis. During this metabolic turnover only the phosphorylinositol portion of the molecule is renewed, as expected if hydrolysis occurs by the action of a phospholipase C, such as phosphatidylinositol phosphodiesterase. Thus, these data suggest that agents which produce in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers lead to a reduction in either the amount or the activity of phospholipase C.     

Gel electrophoretic analysis of histones in lens epithelium, lens fiber, liver, brain, and erythrocytes of late chick embryos.

Histones from 19-day-old chick embryo lens epithelium, lens fibers, liver, brain, and erythrocytes were electrophoresed in polyacrylamide gels using buffers containing sodium dodecylsulfate, acetic acid urea, or mixtures of Triton X-100 acetic acid urea. In the last two buffer systems, histone bands were characterized by their apparent molecular weights determined by electrophoresis in the second dimension in sodium dodecylsulfate containing polyacrylamide gels. From the densitograms of the stained gels, the relative proportion of protein in different histone bands was estimated. With the exception of the erythrocyte-specific histone H5, all histones from different tissues examined at any of the gel systems migrated with the same mobilities. In lens epithelium and lens fibers, all histones were present in identical proportions. As compared to liver and brain, the total amount of histone Hl was significantly lower in lens cells and erythrocytes, possibly reflecting differences between the differentiated states. However, no tissue-specific differences were found in the relative distribution of histone Hl I and Hl II among lens epithelium, lens fiber, liver and, brain, but a threefold higher Hl I : Hl II ratio (0.5--0.7) was found in erythrocytes.     

Clonal analysis of vertebrate myogenesis. II. Environmental influences upon human muscle differentiation

In vitro procedures for obtaining the differentiation of human fetal muscle colonies were developed, and the sensitivity of clonal differentiation to environmental influences was examined. Human muscle colonies are capable of differentiating in the absence of an exogenous collagen substrate. The dependence of clonal diffeentiation upon the addition of chick embryo extract to the culture medium is determined by the serum type used in the medium and by the substrate upon which the colonies are grown. Clonal differentiation also depends upon conditioning of the medium by the colonies. The rate of medium conditioning is affected by clonal density and initial medium composition. The required medium modification is not species specific since medium conditioned by chick muscle cells also permits the early differentiation of human muscle clones. By manipulating the various environmental parameters described above it has been possible to define a number of in vitro conditions which permit a normal rate of cell proliferation but do not permit cell fusion. Results from these experiments are discussed in terms of their developmental implications.     

Gel Electrophoretic Analysis of Histones in Lens Epithelium, Lens Fiber, Liver, Brain, and Erythrocytes of Late Chick Embryos

Histones from 19-day-old chick embryo lens epithelium, lens fibers, liver, brain, and erythrocytes were electrophoresed in polyacrylamide gels using buffers containing sodium dodecylsulfate, acetic acid urea, or mixtures of Triton X-100 acetic acid urea. In the last two buffer systems, histone bands were characterized by their apparent molecular weights determined by electrophoresis in the second dimension in sodium dodecylsulfate containing polyacrylamide gels. From the densitograms of the stained gels, the relative proportion of protein in different histone bands was estimated. With the exception of the erythrocyte-specific histone H5, all histones from different tissues examined in any of the gel systems migrated with the same mobilities. In lens epithelium and lens fibers, all histones were present in identical proportions. As compared to liver and brain, the total amount of histone H1 was significantly lower in lens cells and erythrocytes, possibly reflecting differences between the differentiated states. However, no tissue-specific differences were found in the relative distribution of histone H1 I and H1 II among lens epithelium, lens fiber, liver and, brain, but a threefold higher H1 I: H1 II ratio (0.5–0.7) was found in erythrocytes.