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”

DEVELOPMENTAL

BIOLOGY

115,353-362

(1986)

In Vitro Analysis of Cellular Metaplasia from Pigmented Epithelial Cells to Lens Phenotypes: A Unique Model System for Studying Cellular and Molecular Mechanisms of ‘ ‘Transdiff erentiation” YOSHIAKI

*Laboratory

of Biology, Aichi of Developmental Biology,

Medical National

Received

University, Institute

August

ITOH*

AND

GORO

EGUCHI?’

Nagakute, Aichi 480-11 and tDivision of Morphogenesis, Basic Biology, Nishigonaka 38, Myodaijicho, Okazaki

for

8, 1985; accepted

in revised

form December

Department 4.44, Japan

12, 1985

Pigmented epithelial cells (PECs) were dissociated from eyes of S- to g-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 b-erystallin 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. 0 1986 Academic press, IX.

INTRODUCTION

Transdifferentiation of pigmented epithelial cells (PECs) into lens cells is a clear example of cellular metaplasia in which a once-specialized cell type can switch its differentiation to another (For review see, Clayton, 1978,1979,1982; Eguchi, 19’76,1979,1983; Okada, 1980, 1983; Reyer, 1977; Scheib, 1965; Yamada, 1977, 1982). Since Eguchi and Okada (1973) first used clonal cultures to demonstrate that the progeny of PECs dissociated from 8+-day-old chick embryos can switch phenotype to become lens cells, sufficient information obtained through similar studies has been accumulated to suggest that such a dormant ability to transdifferentiate into lens cells might be widely conserved in vertebrate PECs (Eguchi, 1979; Eguchi and Okada, 1973; Eguchi et al., 1974; Yasuda et al., 1978). Therefore, an in vitro system of PECs would be a useful experimental model for analyzing mechanisms which control transdifferentiation at both cellular and molecular levels. Unfortunately, the efficiency of in vitro transdifferentiation of PECs into lens phenotype has been low and prolonged periods of cultivation are required for transdifferentiation to occur. We have established a useful and efficient in vitro system in which transdifferentiation of PECs can be easily regulated and in which homogeneous population of cells i To whom

reprint

requests

should

be addressed.

353

can be obtained at various states during transdifferentiation. Phenylthiourea (PTU), a potent inhibitor of melanogenesis, enhances transdifferentiation of PECs into lens phenotype (Eguchi, 1976; Eguchi and Itoh, 1981, 1982; Eguchi et al, 1982; Itoh and Eguchi, 1986). In addition, both testicular hyaluronidase (HUase) and ascorbic acid (AsA) promote transdifferentiation of lens cells in cultures of neural retinal cells (Itoh, 1976,1978). In contrast, PECs stably maintain their differentiated state when cultured on artificial collagen substrata (Eguchi, 1979; Yasuda, 1979). In this paper, we report that the dedifferentiated PECs (dePECs) with multipotential for differentiation can be established in cultures by introducing dFBS, PTU, HUase, and ascorbic acid to the culture medium and that these dePECs can readily express either lens or pigment cell specificities depending upon their culture conditions. The system reported in this paper is able to provide a homogeneous cell population at each step of transdifferentiation from PECs to lens cell phenotype which permits critical analysis of molecular mechanisms involved in each step of transdifferentiation. MATERIALS

AND

METHODS

Preparatim of cells. Eight- to nine-day-old chick embryos were used as sources of PECs. Pure pigmented epithelia were dissected from posterior halves of eyes, by treatment with 0.05% EDTA in Ca2+- and Me-free 0012-1606/86 $3.00 Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

phosphate-buffered saline (CMF-PBS) for 30 to 50 min at room temperature. Isolated epithelia were then incubated in 0.1 to 0.2% trypsin in CMF-PBS for 10 min at 37°C (Eguchi and Okada, 1973). Culture media. The following culture media were used: (a) EdF medium: This served as the basic culture medium by supplementing Eagle’s MEM (Nissui Co., Tokyo) with 6 to 8% dFBS (fetal bovine serum dialyzed against Hanks’ saline), 2 mM L-glutamine, and 26 mM sodium bicarbonate. (b) EdFA medium: This medium was EdF medium containing 0.15 mM ascorbic acid and was used for maintenance of PECs dissociated freshly from chick embryos in primary culture and also for initiating of redifferentiation of dePECs. (c) EdFPH medium: The medium was used to permit PECs to dedifferentiate. EdF medium was supplemented with 0.5 mMphenylthiourea (Wako Pure Chemicals Co., Osaka) and testicular hyaluronidase (Boehringer-Mannheim Co., Mannheim; approx 1000 U/mg) at a concentration of 250 U/ml of medium. (d) EdFPHA medium: This medium was prepared by supplementing EdFPH medium with 0.15 mM AsA, and was used for initiating lentoid differentiation in cultures of dePECs. In some of cultures, HUase was supplied at a concentration of 500 U/ml of medium. Procedure for cell culture. About 5 X lo5 PECs freshly dissociated from chick embryos were seeded into a 3.5cm plastic culture dish (Falcon Plastic, No. 3001) with 2 ml of EdFA medium and cultured in a COz-incubator (95% air-5% COB, 100% humidity) with medium replacement every 2 days. When cultures reached confluence, cells were dissociated in the following step to produce dedifferentiated PECs. Confluent primary cultures were washed two times with CMF-PBS; treated with 0.05% EDTA in CMF-PBS for 5 min; and subsequently treated with 0.1 to 0.2% trypsin in CMF-PBS for 10 min at 37°C. After a wash with EdFPH medium, 1 X lo5 dissociated PECs were seeded into a 3.5-cm dish (in 2 ml of EdFPH medium) and were maintained with medium replacement every 24 hr. Before cultures reached confluence (around Day 10 after initiation of each culture), cells were subcultivated to the next generation. Cells were then maintained for approximately 20 days in EdFPH medium before final transfer into medium which initiated redifferentiation or transdifferentiation. In some particular experiments, high cell density culture up to 5 X lo6 cells per 3.5~cm dish was applied according to their purpose. The total number of cells in a dish was determined at 24 hr following inoculation and every 5 days thereafter. The growth rate was estimated by calculating the average number of cells per dish, using two randomly selected culture dishes. Identi$cation of diferentiated properties. Cultures in which pigment cells or lens cells differentiated were dis-

VOLUME 115,1986

sociated as described previously to identify differentiated phenotypes and to estimate the proportion of differentiated cells in each culture. In addition, we monitored the appearance of b-crystallin as a marker of differentiation of lens cell phenotype, whereas differentiated pigment cells were detected by presence of melanin. After fixation of dispersed cells with absolute ethanol at -2O”C, smear preparations on glass microscope slides were immunohistochemically stained with monospecific anti-&crystallin antiserum and FITC-conjugated antirabbit goat antiserum to detect lens cells, and with 5% AgNOa (formula of Fontana, according to Lison, 1960) to detect pigmented cells. Measurement of melanin content. Cultures in which pigmented cell differentiation was initiated were harvested and dissociated every 5 days. Melanin content of these cultured cells was estimated by photometrical measurement of cell lysates according to the method of Oikawa and Nakayasu (1975). Measurement of d-crystallin content. Relative analysis for the efficiency of transdifferentiation of cultured PECs into the lens phenotype was made by counting the number of all discrete lentoid bodies developed in an entire dish. This method was based on the observation by Araki and Okada (1977) that the amount of 6-crystallin is closely correlated with the frequency of lentoid body formation in cultures of neural retinal cells. The amount of d-crystallin produced by cultures was also measured every 5 days after initiation of lentoid differentiations by Laurell’s method for quantitative immunoelectrophoresis (Laurell, 1966; Okada et al., 1983). RESULTS

Dediflerentiation of PECs and Maintenance of Their Dediferentiated State

Pigmented epithelia reconstructed in vitro by culturing PECs in EdFA medium for 2 weeks were dissociated and transferred to EdFPH medium, to initiate their dedifferentiation. Most PECs thus attached to the plastic substratum, spread, and began to proliferate within 2 days. Since de novo synthesis of melanin was completely suppressed by PTU, PECs gradually lost their differentiated phenotype. Melanosomes in each PEC became too scarce to be detected with the light microscope by the time when cultures attained confluence around 15 days after inoculation. All cells which had reached the dedifferentiated state could be maintained by subsequent culturing in EdFPH medium. These cells expressed vigorous growth potential but no identifiable differentiated properties (Fig. la). Therefore, we regarded them as dedifferentiated PECs (dePECs). When a confluent monolayer was formed,

ITOH

AND

EGUCHI

Bipotent

Dedifferentiated

State

FIG. 1. Phase microscopic images of various differentiative states expressed by chick embryonic PECs depending upon their culture conditions. (a) Actively growing dePECs in a culture on Day 10 after inoculation (X119). (b) DePECs in a confluent culture showing large cytoplasmic processes (arrows in an inset) (X119, inset: X570). (c) Redifferentiated PECs from dePECs. Subconfluent dePECs cultured with EdFPH medium were transferred in EdFA medium and maintained for 12 days (X190). (d) Cell piling and lentoid body development in cultures of dePECs which were maintained in EdFPHA medium. Lentoid bodies (LB) shown in this phase micrograph began to appear in the multilayered regions on Day 12 after seeding (X48).

dePECs were flattened; loosely associated with each other; and exhibited a few large surface processes which differed from the microvilli characteristic of well-differentiated PECs (Fig. lb). Cells expressing the dePEC phenotype usually reached to confluence within 15 days when cultured in EdFPH medium with an inoculum size of 1 X lo5 cells per 3.5cm dish. At this stage, cells did not express any differentiation specificity but did continue to proliferate by loss of contact inhibition of growth. Therefore, dePECs

gradually piled up and formed multicellular layers in many parts of each culture when maintained for a long time in EdFPH medium. When confluent dePECs were dissociated and transferred at low cell density to fresh dishes containing EdFPH medium, they continued to proliferate and eventually formed multicellular layers from which lentoid bodies began to develop as described later. Pure population of dePECs can be maintained for more than 2 months by periodic subcultivations at intervals of approximately 15 days.

356 Expression

DEVELOPMENTAL

of Pigment

Cell Specificity

BIOLOGY

of Lens Cell Spe&ticity

115. 1986

by dePECs

Soon after replacement of EdFPH medium by EdFA medium (standard medium containing 0.15 mMAsA) at the subconfluent stage of dePEC cultures, the large cytoplasmic processes of dePECs (Fig. lb, inset) began to disappear and cells temporarily entered a non-proliferative, quiescent state. Approximately 2 days after replacement of the medium, dePECs started to proliferate. Concomitant with proliferation, the cells came into close contact with each other forming a typical polygonal epithelium (Fig. lc). Pigmented cells began to reappear by the second day of culture in EdFA medium and it was possible to observe pigmentation with the unaided eye by the third day. The relative amount of melanin contained in each culture increased from Day 3 to 15 (Fig. 2). Histochemical examination of smear preparations of dissociated cells revealed that more than 95% were pigmented on Day 20 after culturing. No sign of lens cell phenotypes were detected throughout the redifferentiation of dePECs when cultured in EdFA medium. Expression

VOLUME

by dePECs

Sister populations of dePECs which repigmented in EdFA medium were transferred to EdFPHA medium when cultures reached near confluence. These dePECs continue to proliferate without any characteristic morphological change. Even in the fully confluent state, cells

Days in culture FIG. 2. Graph showing the increase in the relative amount of melanin during redifferentiation of dePECs to PECs, determined by optical absorbance per 1 X 10’ cells. O.D. value for freshly dissociated PECs from 8$-day-old chick embryo was found to correspond to approximately 1.5. A: Cultures with EdFA medium, PH: Cultures with EdFPH medium.

Days in culture FIG. 3. Lentoid differentiation in cultures of dePECs maintained in EdFPHA medium (PHA), EdFPH medium (PH) and EdFA medium (A), respectively. EdFPHA medium effectively stimulated lentoid differentiation of dePECs and no lentoid development was achieved in EdFA medium.

continued to express a high growth rate. Consequently, they eventually piled up to form multicellular layers in many regions of the culture dish. Lentoid bodies consisting of well-differentiated lens fibers (Okada et ak, 1973) began to appear in these multicellular layers around Day 10 after transfer (Fig. Id). The number of discrete lentoid bodies appearing in each 3.5-cm dish rapidly increased, and eventually exceeded 1000 at the final stage of culture (Fig. 3). No differentiation of pigmented cells was observed in these cultures. It is important to note that formation of lentoid bodies was confined to multilayered areas surrounded by densely packed depigmented cells which resembled PECs in shape. These densely packed cells could readily melanize within a few days (mostly within 48 hr) when they were exposed to EdFA medium. This observation strongly suggests that the multilayering of dePECs may be an essential prerequisite for differentiation into lens cells. Amplification of Differentiation to Lens Cells

of dePECs

DePECs dissociated from confluent cultures were seeded in 3.5-cm dishes at different cell density and maintained with EdFPHA medium. When about 2 X lo6 cells were seeded in a dish, most cells attached to the

ITOH

AND

EGUCHI

&potent

plastic substratum and formed a monolayer within 24 hr. When much larger number of dePECs (more than 4 X lo6 cell per dish) were seeded, they could form a multilayered cell sheet as soon as they attached to the plastic substratum. Such a multilayer of dePECs could be maintained with substantial cell growth in EdFPHA medium and exhibited features entirely different from those observed in the monolayer cultures previously described. Lentoid bodies began to appear as early as 5 days after inoculation, whereas at least 10 days were usually required for differentiation of lentoid bodies in monolayer cultures of dePECs in EdFPHA medium (Figs. 4, 5). It should be noted that lentoid bodies thus formed were flattened and often fused with each other, a marked contrast to the spherical lentoid bodies which were formed in monolayer cultures of dePECs in EdFPHA medium (compare Fig. 4 with Id). At the terminal phase of culture, almost the entire area of each dish was occupied by lentoid bodies (Figs. 4b, 5B). Figure 5B shows the results of quantitative measurements of Scrystallin contained in cultures at different stages. d-Crystallin could be detected on Day 5 after transfer and the amount increased markedly until Day 15 in coincidence with the increase in the number of lentoid bodies counted directly in living cultures. In contrast, cultures started with lower cell densities exhibited many epithelial islands, consisting of densely packed cells, and a number of discrete lentoid bodies in more multilayered areas of the culture.

FIG. 4. A phase micrograph showing extensive development (4 X lo6 per 3.5~cm dish) and maintained in EdFPHA medium. lentoid bodies (LB) began to develop synchronously (X38). (b) (LB), which fused each other, have developed throughout the

Dedifferentiated

State

357

Demonstration of Bipotential Nature in Diflerentiation of dePECs

It was important to determine whether dePECs can express both specificities of pigment or lens cells depending upon given culture conditions, since the possibility remained that the dePEC population consisted of at least two different precursor cells of lens and pigment cells. To test this possibility, multilayered cell sheet started from 4 X lo6 cells dissociated from an identical culture line of dePECs were maintained with EdFA medium and EdFPHA medium, respectively, and the frequency of lens or pigmented cells appearing in each culture was estimated at various stages of cultivation. Although none of the dePECs maintained in EdFPH medium reacted with anti-b-crystallin antibody, significant numbers of cells from cultures maintained in EdFPHA medium for 5 days could react with the antibody (Figs. 6,7). These cells rapidly increased in number, resulting in more than 90% of cells becoming cross-reactive by Day 20 of culture. These results show that cells which express lens specificity, determined by presence of d-crystallin, have differentiated from dePECs. However, cells identified as pigment cells could not be found throughout the entire period of culture in EdFPHA medium. In cultures with EdFA medium, pigmented cells became detectable by 2 days after inoculation. Even if started from multilayered sheets of dePECs, pigmented

of lentoid bodies from dePECs, which were seeded at extremely high cell density (a) Initial phase of lentoid differentiation on Day 5 after seeding. Many flattened A typical appearance of cultures around 10 days after seeding. Lentoid bodies culture (X38).

358

DEVELOPMENTAL BIOLOGY

ul .I 2 n

4,000

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: 1

VOLUME 115, 1986

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P 1,000

/”

PH or A I

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0 (A)

Days

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FIG. 5. Amplification of the expression of lens phenotype in cultures started from high cell density and maintained with EdFPHA medium. (A) Lentoid body formation was conspicuously accelerated in the culture with inoculum size of 4 X lo6 dePECs per 3.5-cm dish in comparison with the lower inocula. It was difficult to estimate acculate number of lentoid bodies in a dish from around 10 days after seeding. (B) Result of quantitative analysis of b-crystallin production expressed by dePECs maintained in EdFPHA medium. In this experiment 4 X lo6 dePECs were seeded in a 3.5-cm dish and Gcrystallin contents were determined by Laurell’s quantitative immunoelectrophoresis. A: EdFA medium, PH: EdFPH medium, PHA: EdFPHA medium.

cells rapidly increased in number. More than 95% of cells maintained in these conditions invariably repigmented by Day 20. However, neither lentoid development nor anti-d-crystallin antibody cross-reactive cells could be detected. Parallel with pigmentation, cells eventually reconstituted a typical monolayer of closely packed, polygonal heavily pigmented cells (cf. Fig. lc). Thus, dePECs can be regarded as dedifferentiated PECs with at least two potential pathways for differentiation. Furthermore, the phenotype which develops can be efficiently regulated by artificially manipulating culture conditions (Fig. 8). DISCUSSION

FIG. 6. Transmission photomicrographs (a and b) and fluorescent micrographs (c and d) of smear preparations of dePECs cultured in different conditions. For detecting melanin production (a and b) and &crystallin expression (c and d) samples were stained wit.h AgNOa or with anti-&crystallin antibody and FITC-conjugated second antibody, respectively. (a and c) Dissociated cells in the dedifferentiated state (dePECs) corresponding on Day 0 after culturing. (b) Heavily pigmented cells differentiated from dePECs cultured with EdFA medium for 20 days. (d) Lens cells differentiated from dePECs cultured with EdFPHA medium for 20 days, all cells of which exhibit strong fluorescence. (X150).

Since in vitro transdifferentiation of PECs from chick embryos was demonstrated (Eguchi and Okada, 1973), the cell culture system of chick embryonic PECs has become a useful tool for analyzing the mechanisms involved in the phenomena of cell-type conversion from once-specialized cells to other cell types. Based on cell culture studies relating to transdifferentiation of ocular tissue cells (reviews: Clayton, 1979; Eguchi, 1976, 1979; Eguchi and Itoh, 1981; Okada, 1980; Yamada, 1977), we have paid special attention to environmental factors

ITOH

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10

Days

(A)

Bipotent

ECUCHI

15

Dediflerentiated

20

Days in

(B)

in culture

359

State

culture

FIG. ‘7. Efficiency of transdifferentiation and redifferentiation from dePECs to lens and pigment cell phenotypes, respectively, revealed by smear testing. DePECs were inoculated at a density of 4 X lo6 cell per 3.5~cm dish and maintained in two different media; EdFA medium (@) and EdFPHA medium (0). Smear preparations were prepared at various stages of culture and were stained with AgNOa or with anti-& crystallin antibody and FITC-conjugated second antibody for detection of pigment cells and of lens cells, respectively. The percentage of positive cells was obtained by scanning 1000 cells at each test.

which affect cell surface functions to regulate the transdifferentiation of PECs into lens cells in vitro. Close cell-cell contact might be one of the requisites for stabilization of the differentiated state of PECs in chick both in vitro and in vivo. They stably maintain their differentiated state in a cohesive monolayered cell sheet by means of close intercellular communication and

PTU HUase

Af*i*nfn c?ystallln

Contact

(6) lnblbltlon of growth:

:

-

:

-

adhesion (Eguchi, 19’79; Honda and Eguchi, 1980; Honda et al, 1984; Owaribe et ab, 1981) and of interaction between cells and a collagenous substratum (Eguchi, 1979; Yasuda, 1979). Thus, it is reasonable to hypothesize that close cell-cell and cell-substratum contact, mediated by the cellular microenvironment, is one of the necessary conditions for stabilizing the differentiated state of

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FIG. 8. Schematic representation of the culture system which permits the in vitro transdifferentiation of pigmented epithelial cells from 8- to g-day-old chick embryos. Redifferentiation of dePECs to PECs can be also achieved in the standard medium (EdF medium), although initiation of pigmentation is delayed. PEC: pigmented epithelial cell, dePEC: dedifferentiated pigmented epithelial cell, rePECs: redifferentiated pigmented epithelial cells, tLC: transdifferentiated lens cell, PTU: phenylthiourea (0.5 to 1.0 m&f), HUase: testicular hyaluronidase (250-500 U/ml medium), AsA: ascorbic acid (0.15 mM).

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DEVELOPMENTAL BIOLOGY

PECs. A disruption of normal cell contact has been emphasized as a cue for the conversion of cell phenotype in retinal glia cells also (Moscona and Degenstein, 1981; Moscona et al, 1983). A similar hypothesis can be applied to explain the different potentiality in differentiation of dorsal and ventral pigmented epithelia in regeneration of extirpated lens in newts (Sato, 1930, 1951; Mikami, 1941; Watanabe, 1978), since the dorsal predominance of pigmented epithelia in lens differentiation in viva completely disappears in cell culture conditions which start from dissociated PECs organizing these epithelia (Eguchi et al, 1974; Abe and Eguchi, 1977). The following observations support this hypothesis. Chick embryo PECs express a tendency to overlap onto the cell sheet presumably by alteration of their surface functions so that lentoid bodies always differentiate in these multilayered areas, when cultured in the presence of PTU which might affect cell surface properties and reduce cell-cell communication (Eguchi and Itoh, 1981, 1982; Eguchi et al., 1982; Itoh and Eguchi, 1981, 1982, 1986; Itoh et al., 1979). Furthermore, the vesicles, which were artificially prepared from a depigmented sheet of PECs and then cultured on soft agar medium containing PTU, readily develop to lens structures with fully differentiated lens fibers surrounded by an epithelium, showing clear contrast to control vesicles (Eguchi and Itoh, 1982; Eguchi et aZ., 1982). Perhaps, in the vesicles cultured on soft agar medium, each PEC is freed from the restriction of cell-substratum adhesion so that PECs readily change their topological position if cell-cell contact is reduced by PTU. To further investigate the hypothesis proposed above, HUase was applied to culture medium to modify the cell surface. Although HUase itself has no effect on transdifferentiation of PECs from older chick embryos, the enzyme was found to amplify the enhancing effect of PTU on the transdifferentiation of PECs into lens cells. Clearly, this enzyme affects microenvironments surrounding PECs cultured in vitro by digesting proteoglycans which may be responsible for the maintenance of normal physiological activity at the cell surface. In fact, we know that the constituents of surface glycoproteins of PECs were significantly altered during dedifferentiation of PECs to dePECs and redifferentiation of dePECs to either lens or pigment cells (Karasawa and Eguchi, 1985). In addition, the effect of PTU as a potent inhibitor of melanogenesis should be noted. This substance completely suppressed melanogenesis in PECs so that they appeared to be dedifferentiating. Although it has been suggested that PTU accelerates the transdifferentiation of PECs by suppressing melanogenesis (Eguchi and Itoh, 1981), that this may not be the case is indicated by more

VOLUME 115, 1986

recent observations (Masuda and Eguchi, 1982, 1984), since it has been known that suppression of melanogenesis by other inhibitor like thiourea does not enhance the transdifferentiation of PECs, as PTU, naphthylthiourea and methylthiourea do. The bipotent nature of dePECs for differentiating to either lens or pigment cells was clearly demonstrated in the present study. None of the dePECs express pigment cell characteristics when cultured with medium permitting them to differentiate to lens cells, whereas almost all sister populations of dePECs, which expressed lens characteristics, readily redifferentiated to pigment cells when cultured with another medium permitting them to differentiate to pigment cells. Thus, we can conclude that the differentiated state of PECs can be modulated and that redifferentiation from such a state can be completely manipulated. Furthermore, we are presently exploring the utility of this system in molecular analyses of the differentiated phenotype using d-crystallin cDNA cloned by Yasuda et al., (1984). A preliminary result indicates that 6-crystallin gene, which is specifically expressed in avian lens cells, is already transcribed in the dePEC but no matured mRNA of Scrystallin can be found (Agata and Eguchi, 1984; Eguchi, 1986). A mode of transcription of some oncogenes such as myc-, myb, and ?-as-genes has been also investigated in the present system (Eguchi, 1986; Eguchi and Agata, 1985). It seems that studies utilizing the present system provides a unique opportunity for analyzing the molecular mechanisms of cellular transdifferentiation and for describing the molecular nature of multipotent, undifferentiated cells with high growth potential in terms of specific gene expression. Finally, we should not overlook the question of what role is played by ascorbic acid and dialyzed fetal bovine serum in our culture system. However, at the present stage of our study, there is insufficient information to permit a clear understanding of the complex interactions which must occur as a result of these elements in our system. In conclusion, we can maintain in vitro bipotent dedifferentiated PECs derived from 8- to g-day-old chick embryos which can be experimentally modulated to a fully differentiated state of either lens or pigment cells by manipulating culture conditions. Further analysis of the molecular basis for transdifferentiation permitted by this system will contribute to understanding the regulation of the differentiated state in both normal and abnormal development, and also in carcinogenesis (neoplasia). We are indebted to Dr. R. Kodama and Dr. K. Agata, Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology and to Dr. Y. H. Itoh, Aichi Medical University

ITOH AND EGUCHI

Bipotent

for their kind assistance in the present study and for useful advice and discussions. We also thank Professor Robert 0. Kelley, University of New Mexico, School of Medicine for his critical reading of the manuscript. This study was supported in part by Grants-in-Aid for Special Project Research (Project 58119005 and 58113007) and for Basic Cancer Research (Project 58010034) from the Ministry of Education, Science and Culture to G.E. and also supported in part by a Grant-inAid for Encouragement for Young Scientists (Project 57740408) from the Ministry of Education, Science and Culture to Y.I. REFERENCES ABE, S., and EGUCHI, G. (1977). An analysis of differentiative capacity of pigmented epithelial cells of adult newt iris in clonal cell culture. Dev. Growth Dger. 19,309-317. AGATA, K., and EGUCHI, G. (1984). &Crystallin gene is transcribed in multipotent dedifferentiated pigmented epithelial cells. Da? Growth fife?. 26,385. [Abstract] ARAKI, M., and OKADA, T. S. (1977). Differentiation of lens and pigment cells in cultures of neural retinal cells of chick embryos. Dev. BioL 60,278-286. CLAYTON, R. M. (1978). Divergence and convergence in lens cell differentiation: Genetic regulation in the vertebrate lens cell differentiation: Regulation of the formation and specific content of lens fiber cells. In “Stem Cells and Tissue Homeostasis” (B. I. Loyd, C. S. Potten, and R. J. Cole, eds.), pp. 115-180. Cambridge Univ. Press, London/New York. CLAYTON, R. M. (1979). Genetic regulation in the vertebrate lens cell. In “Mechanisms of Cell Change” (J. D. Ebert and T. S. Okada, eds.), pp. 129-167. John Wiley and Sons, New York. CLAYTON,R. M. (1982). Cellular and molecular aspects of differentiation and transdifferentiation of ocular tissue in vitro. In “Differentiation In Vitro” British Society for Cell Biology Symposium 4, (M. M. Yeoman and D. E. S. Truman, eds.), pp. 83-120. Cambridge Univ. Press, Cambridge. EGUCHI, G. (1976). “Transdifferentiation” of vertebrate cells in in vitro cell culture. In “Embryogenesis in Mammals” (Ciba foundation Symposium 40), pp. 241-258. Elsevier, Amsterdam. EGUCHI, G. (1979). “Transdifferentiation” in pigmented epithelial cells of vertebrate eyes in vitro. In “Mechanisms of Cell Change” (J. D. Ebert and T. S. Okada, eds.), pp. 273-291. John Wiley and Sons, New York. EGUCHI, G. (1983). The in vitro system of pigmented epithelial cells as a tool for studies of cell differentiation. In “Developmental Biology. An Afro-Asian Perspective” (S. C. Goel and R. Bellairs, eds.), pp. 97-108. Indian Society of Developmental Biologists, Poona. EGUCHI, G. (1986). Instability in cell commitment of vertebrate pigmented epithelial cells and their transdifferentiation into lens cells. In “Instability in Cell Commitment and Transdifferentiation” (T. S. Okada and A. A. Moscona, eds.), Current Topics in Developmental Biology, Vol. 20. pp. 21-37. Academic Press, Orlando, Fla. EGUCHI, G., and AGATA, K. (1985). Transcription of myc-gene is activated in multipotent dedifferentiated pigmented epithelial cells. Dew. Growth D$er. 27,505. [Abstract] EGUCHI, G., and ITOH, Y. (1981). Regulation of differentiation of vertebrate pigmented epithelial cells by microenvironmental factors. In “Pigment Cell 1981: Phenotypic Expression in Pigment Cells” (M. Seiji, ed.), pp. 271-278. Univ. of Tokyo Press, Tokyo. EGUCHI, G., and ITOH, Y. (1982). Regeneration of the lens as a phenomenon of cellular transdifferentation: Regulability of the differentiated state of the vertebrate pigment epithelial cells. Trans. OphthalmoL Sot. UK 102,374-378. EGUCHI, G., and OKADA, T. S. (1973). Differentiation of lens tissue

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