Mitsuko Kosaka Guangwei Sun Masatoshi Haruta and Masayo Takahashi

1. introduction

The discovery of adult stem cells indicated a previously unrecognized degree of plasticity in stem cell function (1-3). Recent extensive studies have suggested that mammalian stem cells residing in one tissue may have the capacity to produce differentiated cell types for other tissues and organs (4-6). However, more recent reports raised questions about some of the earlier results, proposing that transdifferentiation consequent to cell fusion could underlie many observations otherwise attributed to an intrinsic plasticity of tissue stem cells (7,8). Thus, cell transdifferentiation is of great interest, albeit a poorly understood process invoked to explain how tissue-specific adult stem cells can lose their properties and generate new cells of other tissues.

The fact that differentiated adult cells can change their fate has been known for over a century. The phenomenon of Wolffian lens regeneration in newts (9) has attracted the interest of developmental biologists for long time because it is the clearest and most representative example of transdifferentiation naturally occurring in adult vertebrates: Melanin-producing iris pigment epithelial (PE) cells become crystallin-producing lens cells. A number of studies on the phenomenon of newt lens regeneration were published (10-17), but the molecular basis of this switch in the phenotype of PE cells is mostly unknown. At present, revisiting and rethinking the old phenomenon of Wolffian lens regeneration in adult newts could provide a useful opportunity for obtaining a real idea of somatic cell plasticity in vertebrates.

In this chapter, the historical background of the studies on transdifferentiation using PE cells is briefly reviewed. Current knowledge about the differentiation potency of iris PE cells in postnatal and adult vertebrates, including mammals, is summarized.

From: Adult Stem Cells Edited by: K. Turksen © Humana Press Inc., Totowa, NJ

REGENERATION OF LENS

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REGENERATION OF LENS

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REGENERATION OF NEURAL RETINA

epithelium pigmented epilheliunn

Fig. 1. Regeneration of lens and neural retina in the newt. The newt has a strong ability to regenerate lost parts of the body even after the individual has grown into an adult. In addition to the regeneration of limbs, remarkable examples are found in the eye. When the lens is surgically removed through an opening in the cornea, cells of the pigmented epithelium of the iris become depigmented and proliferate to make a new lens, which grows into the size of an adult lens with a morphology indistinguishable from the normal one. When the neural retina is removed, the retinal PE cells proliferate and regenerate a complete retina.

epithelium pigmented epilheliunn

Fig. 1. Regeneration of lens and neural retina in the newt. The newt has a strong ability to regenerate lost parts of the body even after the individual has grown into an adult. In addition to the regeneration of limbs, remarkable examples are found in the eye. When the lens is surgically removed through an opening in the cornea, cells of the pigmented epithelium of the iris become depigmented and proliferate to make a new lens, which grows into the size of an adult lens with a morphology indistinguishable from the normal one. When the neural retina is removed, the retinal PE cells proliferate and regenerate a complete retina.

2. transdifferentiation into lens cells 2.1. Lens Regeneration in Urodeles

The newt has a strong ability to regenerate lost parts of the body throughout its lifetime. In addition to the regeneration of limbs, remarkable examples are found in the eye. When the lens is surgically removed from an adult newt eye, a structurally and functionally complete lens always regenerates from the PE of the dorsal papillary margin of the iris (Fig. 1). This case clearly demonstrates that fully differentiated cells can switch their type of differentiation and be reprogrammed into another differentiative pathway.

Cell culture studies showed that both dorsal and ventral dissociated newt iris PE cells can transdifferentiate into lens cells in vitro (18,19). In addition to iris PE cells, it was found that retinal PE cells of adult newt can transdifferentiate into lens cells in vitro (20).

Through studies to clarify the cellular origin of lens regeneration in the newt, the retinal PE cells of many vertebrate species have been shown to possess a dormant potency to transdifferentiation into the lens (13,14). Recent observations proposed that retinal PE cells isolated even from adult human cells could also transdifferentiate into lenslike cells in vitro (21).

2.2. A Model System of Lens Transdifferentiation Using Chick Retinal PE Cells

Cell culture of PE cells was attempted as a modern approach to establish an experimental system and to analyze transdifferentiation at the cellular and molecular levels. The introduction of phenylthiourea (PTU) and hyalu-ronidase (HUase) in PE cell cultures has permitted exact control of lens transdifferentiation of PE cells (22). When retinal PE cells from chick embryos (E9) were dissociated and cultured in standard medium containing dialyzed FCS, PTU, and testicular HUase, they dedifferentiated rapidly and grew vigorously. By frequent passage before reaching confluence, it is possible to maintain the undifferentiated state in which cells express neither PE type- nor lens type-specific cell markers. Interestingly, the dedifferentiated PE cells can rapidly reexpress the differentiated PE cell phenotype after withdrawal of PTU and HUase. Furthermore, when seeded at high cell density in medium with dialyzed FBS, PTU, HUase, and ascorbic acid, the dedifferentiated PE cells transdifferentiated into lens cells.

Using this unique system of retinal PE cells, the biochemical and molecular studies have been extensively demonstrated and the results summarized (13,14,23). It was shown that the effect of crude HUase on transdifferentiation was because of fetal growth factor 2 (FGF-2) contamination in the commercial preparations of the enzyme, and that FGF-2 promoted growth and lens transdifferentiation of retinal PE cells. In addition, the cell-cell contact and cell-substratum interactions were suggested to be important for the stabilization of retinal PE cells.

Although valuable observations were obtained from the culture system of embryonic retinal PE cells, the attempts to elucidate the molecular mechanisms of transdifferentiation in vitro have met with limited success. During long-term experience with their cell cultures, it was revealed that retinal PE cells from chick embryos were sensitive to culture conditions. In addition, utilization of the chemical agent PTU, an inhibitor of melanin synthesis that modifies cell surface properties (22,24), has made it difficult to analyze lens transdifferentiation systematically. It remains unclear whether the PE cells can be induced to dedifferentiate without PTU. Moreover, it is still obscure whether the undifferentiated state is necessary for lens transdifferentiation to occur in vitro. So, trials for the establishment of an improved culture system led to the use of newt eye iris PE cells, in which lens transdifferentiation naturally occurs.

It has been difficult to prepare a pure culture of the iris PE cells of the chick embryo because of the tight adhesion of the epithelium with the stroma of the iris; therefore, transdifferentiation of the iris PE cells has scarcely been studied. A simple method was established to prepare pure iris PE cells from postnatal chick (25). Interestingly, the iris PE cells isolated from postnatal chicks were much more stably maintained than the retinal PE cells, and their cells transdifferentiated and dedifferentiated efficiently and repro-ducibly through methods similar to those utilized for the chick retinal PE cells (25).

2.3. Analysis for Lens Transdifferentiation Using Iris PE Cells of Postnatal Chick

In an effort to delineate regulatory factors in lens trandifferentiation of iris PE cells, the effects of known growth factors on iris PE cells were tested. FGF-2 was shown to promote cell proliferation and transdifferentiation of iris PE cells similar to retinal PE cells (25). Further analysis showed epidermal growth factor (EGF) also had a significant effect on iris PE cells, just like FGF-2; the addition of EGF and FGF-2 to pure cell cultures of the iris PE cells synergistically induced the phenotypic change of lens transdifferentiation (26). This finding contributes greatly to the simplification of the humoral requirements for the induction of lens transdifferentiation and provides a powerful system for the molecular analysis of lens transdifferentiation. Furthermore, it was found that the addition of EGF alone as well as FGF-2 alone could also induce in vitro lens transdifferentiation of embryonic retinal PE cells from early chick embryos (E5) (Fig. 2).

Mitogen-activated protein (MAP) kinase is the central component of a signal transduction pathway that is activated by growth factors interacting

Fig. 2. Lens transdifferentiation induced with FGF-2 or EGF in embryonic retinal PE cells (25 d in vitro): (A) without added growth factors; (B) grown in the presence of EGF; (C) grown in the presence of FGF-2; (D) grown in the presence of EGF plus FGF-2; (E) with PD098059 for 1 h before the addition of EGF plus FGF-2. Scale bar, 200 pm.

Fig. 2. Lens transdifferentiation induced with FGF-2 or EGF in embryonic retinal PE cells (25 d in vitro): (A) without added growth factors; (B) grown in the presence of EGF; (C) grown in the presence of FGF-2; (D) grown in the presence of EGF plus FGF-2; (E) with PD098059 for 1 h before the addition of EGF plus FGF-2. Scale bar, 200 pm.

with receptors that have protein tyrosine kinase activity. To evaluate the specific role of the MEK (MAP kinase kinase)-MAP kinase pathway in the transdifferentiative action of EGF and FGF-2, PD098059, a specific inhibitor of MEK, was used in culture. The compound completely blocked the dedifferentiation, rather than the transdifferentiation, of PE cells, providing the first evidence of the requirement of MAP kinase pathway activated by EGF and FGF-2 in the early process of lens transdifferentiation (Fig. 2).

2.4. Lens Regeneration and Lens Development

Eye development in vertebrates has been an excellent model system to investigate fundamental processes in developmental biology, from tissue induction to the formation of highly specialized structures such as the lens and the retina. This complex system develops primarily from three embryonic parts: the optic vesicle (OV), which is a lateral evagination from the wall of the diencephalons; the surrounding mesenchyme; and the overlaying surface ectoderm (SE). The OV contacts the SE and triggers a response that leads to a thickening of the SE and triggers a response that leads to a thickening of the ectoderm, the lens placode, which later develops into the mature lens. The lens placode internalizes to form the lens vesicle; the distal OV invaginates to form the optic cup, with the inner layer developing into the neuroretina, and the outer layer forming the retinal PE.

The expression and function of numerous genes have been correlated with defined cell types and stages of eye development. In particular, the study of the transcription factor Pax6 promoted understanding of eye development. Pax6 is a member of the Pax family of transcription factors. It contains two DNA-binding motifs: the paired domain and paired-type homeodomain (27).

Reports have described the temporal and spatial functions of the transcription factor Pax6 in the developing vertebrate eye. Pax6 is shown to play essential roles in successive steps triggering lens differentiation, and in the retina, it junctions to maintain multipotency and proliferation of retinal progenitor cells (28,29).

It was tested whether the regulatory genes in eye development similarly perform an important function during the lens regeneration process through trandifferentiation of iris PE cells. RNA blot analysis has shown that the transcription of the pax6 gene was rapidly activated on induction of lens transdifferentiation of chick iris PE cells after the addition of EGF and FGF-2 in vitro (26). Furthermore, other regulatory genes in lens development, such as six3 (30) and l-maf (31), were also induced during lens transdifferentiation of iris PE cells in vitro (26). Our data led to the suggestion

Fig. 3. Lens "development" and "regeneration." During early development of the vertebrate eye, when the optic vesicle appears near the surface ectoderm, the ectoderm cells begin to differentiate from the lens. When mesenchymal cells derived from the neural crest cover the optic cup, cells in the posterior pole of the outer layer of the optic cup begin to synthesize melanosomes and differentiate from the PE cells. When the lens is surgically removed from an adult newt eye, a structurally and functionally complete lens always regenerates from the PE of the dorsal papillary margin of the iris.

Fig. 3. Lens "development" and "regeneration." During early development of the vertebrate eye, when the optic vesicle appears near the surface ectoderm, the ectoderm cells begin to differentiate from the lens. When mesenchymal cells derived from the neural crest cover the optic cup, cells in the posterior pole of the outer layer of the optic cup begin to synthesize melanosomes and differentiate from the PE cells. When the lens is surgically removed from an adult newt eye, a structurally and functionally complete lens always regenerates from the PE of the dorsal papillary margin of the iris.

that these genes could be master regulators in lens regeneration and normal lens development in vivo, although the developmental origins of cells forming the lens were clearly distinct from each other (Fig. 3).

Fig. 4. Transcriptional activities of pax6, PE-specific, and lens-specific genes in the process of transdifferentiation. Total RNA was isolated and examined by Northern blot analysis. Change of relative mRNA levels of each gene was shown at each step during the lens transdifferentiation of iris PE cells from postnatal chick. Morphological changes of the cells were illustrated and observed in culture conditions permissive for lens transdifferentiation (bottom). The cells gradually lost their PE phenotype, continued to proliferate through the loss of contact growth inhibition, and formed multicellular layers. Typical lentoid bodies developed in the multi-layered portion.

Fig. 4. Transcriptional activities of pax6, PE-specific, and lens-specific genes in the process of transdifferentiation. Total RNA was isolated and examined by Northern blot analysis. Change of relative mRNA levels of each gene was shown at each step during the lens transdifferentiation of iris PE cells from postnatal chick. Morphological changes of the cells were illustrated and observed in culture conditions permissive for lens transdifferentiation (bottom). The cells gradually lost their PE phenotype, continued to proliferate through the loss of contact growth inhibition, and formed multicellular layers. Typical lentoid bodies developed in the multi-layered portion.

2.5. Gene Expressions During Lens Transdifferentiation of Iris PE Cells In Vitro

In the process of lens transdifferentiation, expression of PE-specific or lens-specific genes for differentiation markers is strictly regulated at the transcriptional level (26,32). When iris PE cells could give rise to additional PE cells in control growth medium with serum, the transcripts of PE-specific genes were easily detected, and those of lens-specific genes were absent. In contrast, when the cells were maintained for about 3 wk in conditions permissive for lens transdifferentiation, the transcripts of PE-specific marker genes were not detected; however, the expression of lens-specific crystallin genes were observed (Fig. 4).

The transcript of the pax6 gene was detected at a low level in iris PE cells in control growth medium. After the addition of EGF and FGF-2 in conditions permissive for lens trandifferentiation, the expression of the pax6 gene was rapidly upregulated. Expression of the pax6 gene in the process of lens transdifferentiation was analyzed by RNA blot. The elevated expression of the pax6 gene and the induction of 8-crystallin gene expression were similarly observed in the early stage of lens transdifferentiation, when the levels of other differentiation marker genes hardly changed (Fig. 4). This result suggests that the state of dedifferentiated PE cells expressing no differentiation markers is not necessary for the transdifferentiation process.

8-Crystallin is the major lens protein in the chick and appears first in the lens placode in chick embryos. Recent findings showed that Pax6 binds cooperatively with another transcriptional factor, Sox2, to the 8-crystallin enhancer, forming a ternary complex that mediates 8-crystallin expression in the lens placode (33). During the lens transdifferentiation process in culture, similar interactions may occur in the iris PE cells as in the lens placode during development. This culture system could provide a model system for further functional studies to determine the roles of various important genes in triggering lens differentiation regulatory interactions.

3. transdifferentiation into neuronal cells

3.1. Transdifferentiation of Retinal PE Cells Into Neuronal Cells

In the newt, the retinal PE has retained the capacity to form a new and complete neural retina, including a new optic nerve (Fig. 1). In anuran species, retinal regeneration after complete retinectomy has not been observed (34,35). It is known, however, that transplantation of retinal PE sheets into the posterior eye chamber of Rana or Xenopus leads to the production of a new retina through the transdifferentiation of the retinal PE. Furthermore, the embryonic retinal PE of many species of vertebrate can also be induced to dedifferentiate and transdifferentiate by altering its environment. In birds and mammals, this retinal transdifferentiation of the retinal PE can occur (only over a narrow period during early eye development) in fetal or embryonic stages, but this capacity is lost during development (36). Thus, it has been thought that the ability of retinal PE cells to produce retinal neurons decreases as embryonic development proceeds.

3.2. Multipotentiality in the Iris PE Cells of Postnatal Chicks

As mentioned, dissociated iris PE cells from the postnatal chick can be expanded and transdifferentiate into lens cells in culture. It is unknown whether the iris PE cells can transdifferentiate into neuronal cell types just as embryonic retinal PE cells can. Our observations have shown that the iris PE isolated from postnatal chick cells can be induced into neuronal cells expressing panneural, glial, and specific retinal neuron markers under certain culture conditions (G. W. Sun and M. Kosaka, unpublished data). These results raise the possibility that some iris PE cells from the postnatal chick have the capacity to transdifferentiate into multiple cell types (not only lens, but also retinal neurons) in vitro. Furthermore, our preliminary observations suggested that the postnatal chick iris PE cells retain a population of neural stem cells similar to that found in the embryonic eye (G. W. Sun and M. Kosaka, unpublished data). Taken together, it can be concluded that fully differentiated iris PE cells from the postnatal chick have the ability to transdifferentiate into multiple cell types, which was classically observed in newts.

3.3. Transdifferentiation Into Neuronal Cells in Mammalian Iris PE Cells

In contrast to the data on neuronal transdifferentiation of retinal PE in amphibians, fish, and birds, very few studies have been carried out with mammalian PE. The embryonic rat retinal PE could transdifferentiate into neuronal cells, but this could occur only during a narrow period of development (E12-E13) (36,37).

To know whether the fully differentiated adult mammalian iris PE cells possess any ability to transdifferentiate into neuronal cell types, we purified and cultured iris tissue cells from adult rats. The results demonstrated that adult iris-derived cells generate neuronal cells expressing panneural marker proteins, but not specific markers for retinal neurons as in response to treatment with FGF-2 (38).

As mentioned, it is proposed that regulatory factors in the developmental process may also play an important role for the transdifferentiation of other cell types. Crx is the homeobox gene specifically expressed in the photoreceptors of the mature retina and is crucial in photoreceptor differentiation. Crx binds to and transactivates genes for several photoreceptor cell-specific proteins (39,40).

In an attempt to obtain photoreceptor cells from adult iris cells, it was examined whether iris-derived cells from adult rats could acquire photore-ceptor-specific phenotypes as a result of the ectopic expression of Crx using replication-defective recombinant virus vectors. The iris-derived cells infected with Crx became small and round, characteristic of the rod photoreceptors in monolayer culture, and most of the cells expressed rhodopsin protein, a specific marker of photoreceptors (Fig. 5). In addition, preliminary

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