Transdifferentiation Of Epithelial Cells Research Articles

TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors.

The secreted polypeptide transforming growth factor-beta (TGF-beta) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-beta signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-beta is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma. In the present study, we investigated the effects of TGF-beta on mouse mammary epithelial cells in vitro. TGF-beta reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E-cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event. Since NMuMG cells make high levels of the type I TGF-beta receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-beta responsiveness, we evaluated the role of the Tsk7L receptor in TGF-beta-mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-beta, yet still displayed some TGF-beta-mediated responses. We conclude that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-beta-induced epithelial to mesenchymal transition.

Basic fibroblast growth factor as one of the essential factors regulating lens transdifferentiation of pigmented epithelial cells.

In vitro transdifferentiation of retinal pigmented epithelial cells of the chick embryo into lens cells can be markedly enhanced by culture in the presence of testicular hyaluronidase and phenylthiourea. Since the commercial preparations of hyaluronidase that had previously been used were very crude, a search for the actual effective molecule(s) enhancing lens transdifferentiation was conducted. First, we purified the enzyme and tested the effect of the purified hyaluronidase. Highly purified hyaluronidase itself did not enhance lens transdifferentiation. The crude hyaluronidase was then separated according to affinity with heparin, considering the possibility that the fibroblast growth factor (FGF) is contained in the crude hyaluronidase. Transdifferentiation-enhancing activity was detected in the fraction which was bound to heparin and eluted with 2 M NaCl, where no hyaluronate-degrading activity existed. Analysis of the fraction by SDS-PAGE revealed the existence of an 18 kDa protein whose NH2-terminal sequence was identical to that of basic FGF. The basic FGF derived from bovine brain also enhanced lens transdifferentiation of pigmented epithelial cells. These findings suggest that basic FGF must play a major role in enhancing transdifferentiation of pigmented epithelial cells to lens cells.

Transdifferentiation of chicken retinal pigmented epithelial cells in serum-free culture

A serum-free culture of chicken retinal pigmented epithelial cells has been established in order to analyse how cell-substrate interactions or environmental factors affect the process of transdifferentiation into lens cells from pigmented epithelial cells. The serum-free culture medium for chicken pigmented epithelial cells was Eagle's minimum essential medium, supplemented with chicken transferrin, soybean trypsin inhibitor and bovine insulin. Pigmented epithelial cells were able to survive and grow in the medium for longer than 2 weeks. Collagen did not promote initial cell attachment, but this material effectively supports pigmented epithelial cells to organize monolayer structure characteristics to pigmented epithelium in situ in comparison with the plastic substrate of culture dishes. The process of lens transdifferentiation of chicken pigmented epithelial cells in serum-free conditions was also enhanced with the aid of phenylthiourea and testicular hyaluronidase, which had already been known to promote the transdifferentiation of pigmented epithelial cells in the serum-supplemented condition. Typical lentoid bodies were developed after about 2 weeks of serum-free culture. Thus, we can clearly demonstrate that the chicken embryonic pigmented epithelial cells do not always require a full set of serum factors for their transdifferentiation to lens cells in vitro.

Retinal regeneration.

The goal of research on neural regeneration is to restore brain function following injury. To many, this suggests regrowing damaged axons and re-establishing the interrupted pathways. A second, but little studied aspect of brain regeneration, is the replacement of lost neurons. For example, in some animals the neural retina is reconstituted by regenerative neurogenesis following its partial or total destruction. Two separate processes underlying retinal regeneration have been described: transdifferentiation of retinal pigmented epithelial cells into retinal neural progenitors (in adult urodeles, tadpoles, and embryonic chickens), and alteration in the fate of photoreceptor progenitors intrinsic to the retina (in adult fish).

Expression of gene coding for a melanosomal matrix protein transcriptionally regulated in the transdifferentiation of chick embryo pigmented epithelial cells

Chicken 115-kDa melanosomal matrix protein (MMP115) was purified from cultured pigmented epithelia cells (PECs), and mouse antiserum was raised to isolate cDNA clones. λgt11 expression library made from poly(A) + RNA of the homogeneous population of PECs was screened with the antiserum. Nine positive clones were obtained from 5 × 10 5 independent phages, and inserts of them shared a common nucleotide sequence. The β-galactosidase fusion protein from the longest insert (MM-2, 1.0 kb long) was recognized by the anti-MMP115 antiserum in immunoblotting, and the antibody, which was affinity-selected by the fusion protein, specifically reacted with the 115-kDa protein in PEC extracts. The RNA blot analysis with the MM-2 insert as a probe revealed that a transcript of 2.6 kb was expressed by the PEC in a tissue-specific manner. mRNA expressions in the process of in vitro transdifferentiation from PECs to lens cells were analyzed using the MM-2 insert. The transcripts were detected in neither transdifferentiating, transdifferentiated lens cells nor bipotent dedifferentiated PECs, although the 2.6 kb transcript was vigorously synthesized by redifferentiating into PECs.

The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.

Enhancement of expression of lens phenotype in cultures of pigmented epithelial cells by hyaluronidase in the presence of phenylthiourea

Pigmented epithelial cells isolated from 8–9-day-old chick embryos can transdifferentiate into lens-like cells at the terminal period of the third generation of culture. However, efficiency of this transdifferentiation is usually rather low. Phenylthiourea, a potent inhibitor of melanin synthesis, effectively enhances transdifferentiation of pigmented epithelial cells into lens-like cells in vitro. Lentoid bodies began to appear in the multilayered region of primary cultures of pigmented epithelial cells maintained in medium containing phenylthiourea at concentrations between 0.5 and 1.0 mM. Furthermore, the enhancing effect of phenylthiourea can be amplified with testicular hyaluronidase. Under these conditions, pigmented epithelial cells grow vigorously and lose their differentiative properties, efficiently switching their phenotype into lens-like cells some 20 days after initiation of culture in the presence of both substances. Semiquantitative analysis revealed that testicular hyaluronidase amplified the effect of phenylthiourea more than 100-fold. It has been suggested that phenotypic expression of pigmented epithelial cells during transdifferentiation can be regulated by manipulating the microenvironment in which these cells reside.

Chapter 8 Neural Crest and Thymic Myoid Cells

Publisher Summary Myoid cells commonly appear in the thymus of reptiles, birds, and mammals, including humans. The rather unexpected presence of these cells in this organ has attracted special attention because it has been thought to be related with the autoimmune disease, myasthenia gravis. Most myoid cells are situated in the medulla of the thymus and they share common morphological and functional characteristics with skeletal muscle. The origin of the thymic myoid cells (TMC) has been a matter of dispute and two alternative hypotheses have been proposed. One is that TMC occur as a result of the transdifferentiation of thymic endoderma1 epithelial cells. In the second hypothesis, TMC are considered to originate from the surrounding tissues. Thus, the problem of seemingly ectopic differentiation of myoid cells in thymus is reviewed in this chapter. From the results of electron microscopic and immunohistochemical studies on the chimeric birds, it has been concluded that TMC do not occur by the transdifferentiation of the thymic epithelial cells, but are derived from the neural crest.

Transdifferentiation of Adult Frog Iris in Retina or Lens by Exogeneous Influences.

The mechanisms of transdifferentiation of iris epithelial cells of Rana temporaria (Anura) in culture depending on influences from different sources were studied. In terminally differentiated iris cells, the process of transdifferentiation is initiated by dedifferentiation. Melanosomes are shed from iris cells due to cell surface activity. After depigmentation, iris epithelial cells become capable of proliferating and competent to react to the influences of various exogenous factors. Under the influence of retinal factors secreted by lentectomized tadpole eyes, both dorsal and ventral irises are converted to neural retina. Under the influence of factors from eye vesicles, the irises are converted to neural retina as well. Similar results were obtained in transfilter experiments, in which a 3-day period of transfilter interaction between the irises and eye vesicles ensured depigmentation of the iris followed by transdifferentiation into complete NR with visual receptor. Lentoid formation occurred under the influence of adult frog lens epithelium. Immunofluorescent analysis confirmed the lens nature of the lentoids. In control experiments under the conditions of the tadpole eye orbit, in which programming influences were absent, iris epithelial cells remained unaffected. The problem of true cell-reprogramming to new differentiation in contrast to expression of inherent properties of the iris epithelial cells is discussed.

An electron microscopic study of early developmental stages, myogenesis, oogenesis and cnidogenesis in the anthomedusa, Podocoryne carnea M. Sars.

This descriptive electron microscopic study of the blastogenetic medusa development of Podocoryne carnea focuses on the earliest stages of primordium formation, myogenesis, oogenesis and cnidogenesis. The events which take place at the cellular level prior to the formation of stage 1 (Frey, '68), which are characterized by ecto- and entodermal accumulations of undifferentiated I-cells and the beginning of transdifferentiation of epithelial cells, have been subdivided into four distinct stages (U1-U4). The genealogy of cells participating in medusa differentiation indicates that some cell types of the medusa are derived by transdifferentiation from the polyp's epithelial cells, while others originate by differentiation from I-cells. The myogenesis of the subumbrellar muscle cells resembles vertebrate myocardiac differentiation in many respects.