In order to study the effect of glucose on the differentiation of cultured human colon cancer cells, a subpopulation of HT-29 cells was selected for its capacity to grow in the total absence of sugar. These cells (Glc-cells) exhibit, after confluency, an enterocytic differentiation, in contrast to cells grown with glucose (Glc+ cells), which always remain undifferentiated. The differentiation is characterized by a polarization of the cell layer with apical brush borders and tight junctions, and by the presence of sucrase-isomaltase. The differentiation of Glc- cells is reversible: the addition of glucose to postconfluent cultures of Glc- cells results in an inhibiting effect on the expression of sucrase-isomaltase; switching growing cultures of Glc- cells to the Glc+ medium for several passages results in a progressive reversion to the undifferentiated state, which is completed after seven passages. The dedifferentiation process is associated with a parallel, passage-related, increase in the rates of glucose consumption and lactic acid production, and decreases of intracellular glycogen content, which return to the values of the undifferentiated original Glc+ cells. The values of these metabolic parameters are correlated, at each passage, with the degree of dedifferentiation of the cells. When these dedifferentiated cells, after having been cultured in Glc+ medium for 20 passages, are switched back to the Glc- medium, they readily grow without mortality, and reexpress the same enterocytic differentiation as the parent Glc- cells. These results show that the capacity of this subpopulation to grow and differentiate in the absence of sugar is a stable characteristic. They further suggest that glucose metabolism interferes with the program of differentiation of HT-29 cells.

Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose

Cell aging in vivo and in vitro

Publisher Summary This chapter describes the proliferation and differentiation of retinal pigment epithelium (RPE) during development and regeneration. RPE is a pigmented monolayer of hexagonal cells of neural origin located between the neural retina and the choroid. It is one of the most important structures in the visual system because of the role it plays in the viability and function of the neural retina. The comparison of RPE development in three species (rats, chicks, and humans) showed that the pattern and schedule of RPE growth and differentiation causally related to cell proliferation are significantly different. The chapter discusses the proliferation of pigment epithelium cells in the amphibian retina and iris in the process of retinal and lens regeneration. The importance of analyzing the proliferative activity of pigmented epithelia of the amphibian eye is because of the amphibian's striking potential for the regeneration of the neural retina and lens. Cell proliferation is not only closely related to cell multiplication and the reduction of cell volume but is also essential for RPE melanotic differentiation and for transdifferentiation into other cell types.

Retinal pigment epithelium: proliferation and differentiation during development and regeneration.

The wing and wing-associated muscles of the shoulder and thorax in the bird all cleavefrom common myogenic masses in the developing wing bud and are referred to collectively as brachial muscles. In this study the precise embryonic origin of the brachial muscles was determined using chick-quail chimaeras. Such chimaeras consisted of a graft of one somite taken from a 2-day quail donor embryo transplanted to the equivalent location in a 2-day chick host embryo. The chimaeras were analysed at 9·5–10·0 days in ovo to determine the location of the grafted cells and therefore the structures that were derived from the transplanted somite. The somites that were studied in this manner were somites 13 to 23 inclusive. The results show that only somites 16 to 21 inclusive contribute cells to the brachial musculature; moreover, the cells from a given somite are not distributed randomly among the brachial muscles but populate specific muscles only: thus it has been possible to map the somitic origin of individual brachial muscles. Moreover, there is an indication that each somite plays a unique role in the development of the brachial muscles.

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Brachial muscles in the chick embryo: the fate of individual somites.

Heterogeneity of expression for a variety of characteristics is found among malignant cells in the organism and in culture. Normal cells are relatively uniform when organized in a tissue, but become heterogeneous for many characteristics when they are dispersed and grown in monolayer culture. The heterogenizing effect of growth in culture indicates that the morphology and behavior of normal cells is ordered by their topological relations in tissues and other homeostatic influences of the organism. Weakening of these ordering relations may contribute to malignant transformation, as it usually does in rodent cell culture. Although phenotypic differences among cells of a given type may be transient, they can be perpetuated by protracted exposure to selective conditions. Examples are cited of selection which leads to an adapted state that is heritable for many cell generations after removal of the selective conditions. Such heritable adaptations are analogous to the Dauermodifikationen, or lingering changes, first described in ciliated protozoa and shown there to be under cytoplasmic control. The concept of progressive state selection is introduced to account for heritable adaptation at the cellular level. It depends on the spontaneous occurrence of transient, variant states and their successive selection to progressively higher levels of adaptation to an altered microenvironment. Although the process is basically epigenetic, it may be stabilized by genetic change. The concept is consistent with our present knowledge of tumor development, including progression to metastasis, and with epigenetic aspects of normal development.

The significance of biological heterogeneity.

SUMMARY Embryonic (9-day) chick neuroretinal cells transdifferentiate extensively into lens and pigment cells during prolonged culture (4-5 weeks) in media containing foetal calf serum. Medium conditions which promote the attachment and differentiation of neural cells in other culture systems (e.g. horse serum, high glucose levels) both delay the onset and greatly reduce the extent of transdifferentiation in retinal cultures. In the presence of high glucose, horse serum (but not foetal calf serum) also favours cholinergic neuronal differentiation during the early phases of culture, as shown by the levels of choline acetyltransferase activity and accumulation of labelled choline. Substrate conditions have some effect on cholinergic differentiation (promoted by polylysine-coated dishes) but do not affect later transdifferentiation. These effects may be due in part to selective survival or growth of particular retinal cell types under the various medium conditions tested. Cultures stripped of neuronal cells contain negligible choline acetyltransferase activity, but still transdifferentiate into both lens and pigment cells, although more slowly than control cultures. Cell size distributions reveal a significant depletion of the larger cells in high glucose media with foetal calf serum, but not in those with horse serum.

Influence of serum factors on the prevalence of "normal" and "foreign" differentiation pathways in cultures of chick embryo neuroretinal cells

Embryonic chick neuroretinal cells transdifferentiate into lens cells during culture in media containing foetal calf serum (F). This process is largely inhibited if horse serum plus supplementary glucose (Hg) is substituted for F. This paper explores the effect of medium changeover (from F to Hg or vice versa) on the subsequent appearance of lens-specific δ-crystallin. If cultures are changed from Hg to F up to 12 days of culture, δ-production at 40 days is similar to that for controls maintained in F throughout. Changeovers between 14 and 17 days progressively inhibit subsequent δ production, and after 19 days in Hg, lens transdifferentiation cannot be induced by F. Conversely, if cultures are maintained in F for up to 17 days, a changeover to Hg blocks transdifferentiation, whereas similar transfers performed after 19 days give increased δ production. These results suggest that some retinal cells which will eventually form lens in vitro become so determined between the 12th and 20th days of culture. A mixture of 50% Hg and 50% F medium (FHg) does not support δ production even after 60 days, but in the absence of supplementary glucose (FH), δ appears in considerable amounts by 30 days.

Determination of Chick Neuro‐retinal Cells in Culture: Serum Factors Acting between 12 and 20 Days of Culture Influence the Extent of Subsequent Lens Cell Formation

A switch for transdifferentiation in culture: effects of glucose on cell determination in chick embryo neuroretinal cultures.

Pathways of Differentiation in Chick Embryo Neuroretinal Cultures

Several markers of chick neuroretinal differentiation were monitored in vivo and in culture. All increase markedly between 7 and 20 days of embryonic development in vivo. In vitro , endogenous GABA levels decrease almost immediately, while other neuronal markers increase as in vivo for 2 to 5 days before declining (choline acetyltransferase, acetyl cholinesterase, glutamic acid decarboxylase). Neuronal cell surface markers (binding sites for tetanus toxin, α-bungarotoxin, muscimol), however, reach maximal levels only after 8 days in vitro . Glial markers such as carbonic anhydrase and hydrocortisone-induced glutamine synthetase activities are also expressed only transiently in culture.

M. A. H. Gali

Papers

Influence of serum factors on the prevalence of "normal" and "foreign" differentiation pathways in cultures of chick embryo neuroretinal cells

Determination of Chick Neuro‐retinal Cells in Culture: Serum Factors Acting between 12 and 20 Days of Culture Influence the Extent of Subsequent Lens Cell Formation

A switch for transdifferentiation in culture: effects of glucose on cell determination in chick embryo neuroretinal cultures.

Pathways of Differentiation in Chick Embryo Neuroretinal Cultures

Expression of differentiation markers by chick embryo neuroretinal cells in vivo and in culture