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Keywords:

  • ES cells;
  • transplantation;
  • melanocytes;
  • pigmented epithelium

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Regenerative transplantation of embryonic stem (ES) cell-derived melanocytes into adult tissues, especially skin that includes hair follicles or the hair follicle itself, generally not possible, whereas that of ES cell-derived pigmented epithelium was reported previously. We investigated the in vivo differentiation of these two pigment cell types derived from ES cells after their transfer into the iris. Melanocytes derived from ES cells efficiently integrated into the iris and expanded to fill the stromal layer of the iris, like those prepared from neonatal skin. Transplanted pigmented epithelium from either ES cells or the neonatal eye was also found to be integrated into the iris. Both types of these regenerated pigment cells showed the correct morphology. Regenerated pigment epithelium expressed its functional marker. Functional blocking of signals required for melanocyte development abolished the differentiation of transplanted melanocytes. These results indicate successful in vivo regenerative transfer of pigment cells induced from ES cells in vitro. Developmental Dynamics 237:2394–2404, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Embryonic stem (ES) cells, derived from the inner cell mass of the blastocyst, have the potential to give rise to all cell lineages when introduced into the early embryo. They also differentiate into a variety of cell types in vitro when appropriate environmental cues are reproduced in the culture system (O'Shera,1999; Prelle et al.,2002; Turksen,2002). As ES cells continue to proliferate in the undifferentiated state in vitro, an unlimited source of stem cells or their derivatives may be secured. It is also a potential benefit that ES cells may be genetically manipulated to permit the selective differentiation and/or isolation of a specific cell type.

We previously established culture systems in which melanocytes (Yamane et al.,1999; Aoki et al.,2005; Motohashi et al.,2007) and eye-like structures containing lens, neural retina, a retinal pigmented epithelium (RPE) -like cells (Hirano et al.,2003) could be induced from mouse ES cells. We reported that ES cell-derived RPE cells generated a single-cell layer of mature RPE cells in the developing chick optic vehicle (Aoki et al.,2006). Additionally, there are other reports of the establishment of regenerative therapy models involving the transplantation of fetal or ES cell-derived RPE cells into the subretinal space of animal models of retinal degeneration (Haruta et al.,2004; da Cruz et al.,2007). Partially effective transplantation of fetal human RPE cell sheets into the retinas of patients with retinitis pigmentosa was also reported (Radtke et al.,2002,2004).

Transplantation of cultured or freshly prepared melanocytes into the skin of human vitiligo patients is fairly successful, and re-pigmentation has been accomplished (Olsson and Juhlin,2002; Chen et al.,2004). Similar transplantation of cultured or freshly prepared normal melanocytes or their precursors into mouse skin has been unsuccessful, at least in our hands. Even after the use of Stem Cell Factor (SCF) transgenic mice (Kunisada et al.,1998), which can maintain pigmented melanocytes in their interfollicular skin for their lifetime, we were not successful in transplanting melanocytes into the skin of such mice even after they had been treated with an antagonistic neutralizing antibody for c-kit receptor (ACK2) antibody to eliminate the proliferation of host melanocytes before the transplantation (data not shown). In contrast, we recently showed the successful regenerative differentiation of melanocytes or their precursors prepared from newborn mice when these cells were transplanted into the adult mouse iris (Aoki et al.,2008). In the eye, melanocytes populate the iris stroma, ciliary body, and choroid that are collectively known as the uvea; whereas pigmented epithelial cells form distinctive cell layers in the iris, ciliary body, and retina in a manner independent of melanocytes. Therefore, the iris is composed of a stroma filled with melanocytes and a two-layered pigmented epithelium, which could be a target tissue for the regenerative transfer of both melanocytes and pigmented epithelium. Here, we intended to establish a method for the regenerative transfer of ES cell-derived pigment cells to adult animals for future therapeutic use. Then we tested the induced RPE-like cells and melanocytes for their ability to populate the developed iris tissue.

Our results showed the successful transfer and regenerative differentiation of both pigment cell lineages derived from ES cells in the adult iris tissue, indicating the possible future use of ES cell-derived pigment cells for regenerative medicine. This possibility is especially likely considering the impact of recently established induced pluripotent stem (iPS) cells (Takahashi and Yamanaka,2006; Takahashi et al.,2007; Yu et al.,2007), which are a substitute for ES cells derived from fertilized eggs.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Transplantation of Pigment Cells Prepared From Postnatal Day 0 Mice

To detect control pigment cells from mice for comparison with ES cell-derived pigment cells, we used Dct-lacZ transgenic mice having pigment cell-specific b-galactosidase expression (Mackenzie et al.,1997). In postnatal day (P) 0 mice, both the central region (Fig. 1A) and the peripheral region (Fig. 1B) of the retina contained pigmented epithelium; whereas melanocyte distribution was restricted to the choroids around the anterior rim (where is also called as ciliary marginal zone) in the peripheral region of the retina (Fig. 1B) and optic nerve (data not shown). In P0 skin, migrating melanoblasts were observed in the interfollicular epidermis and hair follicles (Fig. 1F,G). So we used the uveal region of P0 mice excluding the anterior rim and around the optic nerve as a source of pigmented epithelium and P0 trunk skin for melanocytes.

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Figure 1. Comparison of retinal pigmented epithelium (RPE) and melanocytes from postnatal day (P) 0 mice as in vivo counterpart. A,B: LacZ-stained retina from P0 DCT-LacZ mice, showing the location of the pigment cells in the central region between the anterior rim and the optic nerve (A) and the peripheral region adjacent to the anterior rim (B) of the retina. C: Ten days after transplantation of P0 pigmented epithelium. D: Close-up view of the black-pigmented cells with round shape seen in C. E: Hematoxylin and eosin (H&E) -stained section of iris prepared from the host eye seen in C shows the transplanted pigmented epithelium on the IPE side. Black pigmented cells are shown in the insert at the lower left. F: LacZ-stained back skin from P0 DCT-LacZ mice. G: Close-up view of the open box in F shows LacZ-positive melanocytes migrating toward the hair follicle. H: Ten days after transplantation of P0 epidermis. I: Close-up view of the black-pigmented melanocytes with dendritic morphology seen in H. J: H&E-stained iris prepared from the host eye shown in H shows the transplanted melanocytes on the stroma side of the iris. Scale bars = 50 μm.

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Dissociated cells prepared from P0 tissues were transplanted into the adult ICR mouse iris. After 10 days, the transplanted melanocytes spread to a relatively large area of the stromal layer of the iris (Fig. 1H–J); whereas most of the pigmented epithelial cells differentiated around the injection site for transplantation (Fig. 1C–E). Thus, we found the adult iris tissue to be capable of accepting pigment cells, that is, melanocytes derived from skin (Aoki et al.,2008) and retinal pigment epithelium. These results suggest that the adult iris can support the development of both types of transplanted pigment cells, i.e., melanocytes and pigmented epithelial cells.

Transplantation of Melanocytes Differentiated From ES Cells

To investigate the differentiation and integration potential of pigmented cells differentiated from ES cells, we first transplanted ES cell-derived melanocytes. The induction of mature black-pigmented melanocytes with dendritic morphology from undifferentiated ES cells requires a 21-day culture period; and around culture day 17, weakly pigmented dendritic cells appear (Yamane et al.,1999; Aoki et al.,2005, Motohashi et al.,2007). We chose ES cells from 19-day cultures as donor cells for transplantation, because at this time some pigmented melanocytes differentiate from the nonpigmented melanoblasts. The black-pigmented cells allowed us to confirm that the ES cells had successfully generated melanocytes. In our culture system, ES cells differentiated not only into melanocytes (shown in the left column in Fig. 2A–C) but also into neurons (Fig. 2A), glia (Fig. 2B), and smooth muscle cells (Fig. 2C).

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Figure 2. Transplantation of culture day-19 embryonic stem (ES) cell-derived melanocytes. A–C: Immunocytochemistry at 19 days after induction of melanocytes from ES cells on ST2 cells in vitro. Neural (Tuj1), glial (GFAP), and muscle (SMA) cells (shown in center column of photos) as well as melanocytes (shown in the left column; BF indicates the photos taken in the bright field) derived from green fluorescent protein (GFP) -ES cells (shown in the second column from the left) co-exist in our culture system (shown in the right column), as shown in A, B, and C, respectively. Nuclei were visualized by Hoechst staining (shown in the second column from the right). D,E: Ten days after transplantation of culture day-19 ES cell-derived melanocytes into the recipient. D: Brightfield (BF, left) and fluorescent (GFP, right) images of transplant area (arrowheads). E: Brightfield (BF, left) and fluorescent (GFP, right) images of the side opposite to the transplant area. F: Three-micron-thick section prepared from the host eye shown in D. Phase-contrast (PC, upper left) and fluorescence (GFP, lower left) images were merged in the upper right. The black-pigmented melanocytes with their characteristic dendritic shape are GFP-positive transplanted cells. A hematoxylin and eosin (H&E) -stained section is shown at the lower right. G–I: In situ immunohistochemically stained host iris bearing transplanted ES cell derivatives. Whereas all pigmented melanocytes (shown in the left column) express GFP (shown in the second column from the left), the neural (Tuj1), glial (GFAP), and muscle (SMA) cells (shown in the second column from the right) do not express GFP at all (shown in the right column). J,K: Transmission electron microscopic observations of transplanted melanocytes show the typical structures of mature melanocytes, such as numerous pigment granules and melanosomes. Three independent melanosomes including several pigment granules were inserted in the lower left corner in K. Scale bars = 50 μm in A–C,F,G,I,K, 2 μm in J,K.

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When ES cell-derived melanocytes were transplanted into the iris, a large number of them were detected over the surface of the iris (Fig. 2D–F). Most of them were black-pigmented and expressed green fluorescent protein (GFP), and showed a melanocyte-specific dendritic morphology (Fig. 2G–I). They migrated out to the iris region on the opposite side (Fig. 2E) of the transplant site (Fig. 2D, the transplant site shown by arrowheads). After the same sample had been sectioned and stained, the ES cell-derived melanocytes were observed to have spread out in the stromal layer of the iris (Fig. 2F). These transplanted cells contained black pigment granules (shown in the upper left photo in Fig. 2F and the left photo in Fig. 2G–I), and expressed GFP (shown in the lower left photo in Fig. 2F and the second column from the left in Fig. 2G–I) but did not express the neural (Fig. 2G), glial (Fig. 2H) or smooth muscle (Fig. 2I) cell marker used. Black-pigmented cells are terminally differentiated mature melanocytes that do not express proliferation marker gene Ki-67 (Fig. 4J–L, white arrowhead). On the other hand, the unpigmented cells adjacent to these black-pigmented cells seem to be the proliferative melanocyte progenitors expressing Ki-67 (Fig. 4L, black arrowhead). These proliferating melanocyte progenitors were detected in the culture used for transplantation as indicated in the experimental procedures. The culture used for transplantation contained average 0.13% melanocytes and 0.81% Dct-LacZ–positive melanoblasts. Therefore, in approximately 20,000 cells/eye transplanted cells, 30 are matured melanocytes and 170 are melanocyte progenitors or melanoblasts. At 10 day after transplantation, we could detect 1,417 ± 249 blackly pigmented melanocytes in iris (n = 6), clearly indicating the proliferation and maturation of transplanted melanocyte progenitors. By transmission electron microscopy, structures typical of mature melanocytes, such as numerous pigment granules and melanosomes (Fig. 2J,K), were confirmed. These observations show that the grafted ES cell derivatives expressed the properties of melanocyte lineage cells in the recipient adult mouse iris.

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Figure 4. Effect of ACK2 and BQ788 on in vivo development of transplanted embryonic stem (ES) cell-derived melanoblasts. A–D: Day 10 after transplantation of culture day-19 ES cell-derived melanocytes into recipient mouse eyes. At day 2 after transplantation of the melanocytes, phosphate buffered saline (PBS) as a control (A), Ack2 (B), BQ788 (C), or Ack2 and BQ788 (D, shown as “Both”) were administered. Lower magnification view of the whole eye was inserted in the lower left corner of each photo. E–H: Close-up view of the black pigmented cells in A to D. I: The number of mature melanocytes in A to D was counted. Asterisks indicate P < 0.01. J–L: Three-micrometer sections prepared from the host eye. J: Phase-contrast image of black-pigmented melanocytes is shown. K: Immunohistochemically stained host iris at 10 days after transplantation of ES cell derived-melanocytes. A proliferation marker, Ki-67–positive cells are shown (red). L: Merged image of J and K. Not black-pigmented melanocytes but their adjacent unpigmented cells were Ki-67-positive.

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For transplantation, we used unpurified cells prepared from the ES cell cultures, which included neurons, glia, and smooth muscle cells aside from melanocytes, as was shown in Figure 2A–C. Constitutively GFP-positive cells indicate all ES cell derivatives, and most of these GFP-positive cells, were black-pigmented melanocytes with a dendritic morphology (Fig. 2G–I). As mentioned above, the Tuj1-, glial fibrillary acidic protein (GFAP)-, and smooth muscle actin (SMA) -positive cells were not GFP-positive, suggesting that these cells were host-derived cells and had not differentiated from the donor cells. These results indicate the transplanted ES cell-derived melanocyte-lineage cells actually grew and differentiated in the adult iris as did the P0 skin-derived melanocytes.

Transplantation of RPE Cells Derived From Eye-Like Structures Differentiated From ES Cells

Next, we transplanted pigmented epithelium prepared from ES cell-derived eye-like structures. The induction of mature black-pigmented polygonal epithelial cells from undifferentiated ES cells was completed during day 11 of the culture. Around day 9 of ES cultures, a population of weakly pigmented cells appeared surrounding a lens-like transparent, compact cell mass, suggesting the initiation of the maturation of precursors into pigmented epithelial cells, as previously suggested (Hirano et al.,2003; Fig. 3A–C).

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Figure 3. Transplantation of culture day-9 embryonic stem (ES) cell-derived pigmented epithelium present in eye-like structures. A–C: Immunocytochemistry at 9 days after induction of eye-like structures including pigmented epithelium from ES cells cultured on PA6 cells in vitro. Neural (Tuj1) and glial (GFAP) cells (shown in center photos in A and B, respectively) were adjacent to the polygonal pigmented cells (shown in the left column of photos). These pigmented cells expressed RPE65, a retinal pigmented epithelium marker (shown in center photo in C). Pigmented cells (left column) as well as all neural (Tuj1), glial (GFAP), and RPE (RPE65) cells (center column), derived from green fluorescent protein (GFP) -ES cells (second column from the left), co-existed in our culture system (right column) as shown in A, B, and C, respectively. Nuclei were visualized by Hoechst staining (second column from the right). D–F: ES cell-derived pigmented epithelium at 10 days after transplantation into adult mouse iris. E: Close-up view of the boxed area in D. F: Close-up view of the angle area. G,H: Three-micron-thick section prepared from the host eye shown in D. G: A hematoxylin and eosin (H&E) -stained section. H: Phase-contrast image. Black-pigmented epithelial cells with their characteristic round shape are shown. I: In situ immunohistochemically stained host iris at 10 days after transplantation of ES cell derivatives. Black-pigmented epithelial cells with a round shape (left photo) are GFP-positive (second photo from the left) and express RPE65 (center photo). Double-stained images taken in the dark field (second photo from the right) and in the bright field (right) are shown in the left two rows to indicate the GFP-positive ES cell derivatives: RPE65-positive cells and the black-pigmented cells. J,K: Transmission electron microscopic images of the pigment cells show the typical structures of mature pigmented epithelial cells, such as extensive apical microvilli and numerous pigment granules. Scale bars = 50 μm in A–C,G,I, 1 μm in J,K.

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Cells prepared from ES cell-derived eye-like structures including black-pigmented epithelial cells were transplanted into the iris. Ten days later, expansion of pigmented cells was observed on the surface of the iris (Fig. 3D–H), mainly in the vicinity of the transplant area compared with the broader distribution in the case of the transplanted melanocytes. They showed pigmented epithelium-like flat morphology (shown in the left panel in Fig. 3I) and expressed RPE 65, a functional RPE marker (shown in the center photo in Fig. 3I). By using transmission electron microscopy, we observed the transplanted pigmented epithelium to have the typical structures of the mature RPE, such as extensive apical microvilli and numerous, large-sized pigment granules (Fig. 3J,K). Thus, these results would suggest that iris also provides a suitable environment for the regenerative differentiation of pigmented epithelial cells. Thus, these observations would suggest that the iris also provides a suitable environment for the regenerative differentiation of the pigmented epithelial cells. These results indicate the transplanted ES cell-derived pigmented epithelial cells differentiated in the adult iris as did those prepared from the iris of P0 mice.

In Vivo Development of the Transplanted Melanoblasts From ES Cells and the Effect of Antibody Against Kit and Inhibitor of Endothelin 3

To further clarify the normal differentiation of these transplanted cells after transplantation in vivo, especially for the melanocytes which showed their extensive increase in number, we performed functional blocking of signals required for the melanocyte development by using neutralized antibody and/or antagonist after transplantation.

Melanocytes are derived from cells of the closing neural crest that have migrated from the neural tube (Le Douarin et al.,1999); during embryogenesis, melanocytes are keenly dependent on c-kit/SCF signaling. When the proper amount of ACK2, an antagonistic neutralizing antibody against Kit, was administered in utero (Yoshida et al.,1993), all melanocytes disappeared as in the case of Kit mutants such as W/W and W/Wv (Dunn,1937; Russell,1949). EDNRB/Endothelin 3 (EDN3) signaling is also required for the normal development of melanocyte lineage cells, because spontaneous mutants such as piebald-lethal (Ednrbs-l) and lethal spotting (EDN3ls) and those with targeted disruption show almost a completely white coat color (Dunn and Charles,1937; Lane,1966; Hosoda et al.,1994; Baynash et al.,1994). In the induction of melanocytes from ES cells in vitro, these 2 pathways are also important for their development; as the addition of these factors, SCF and/or EDN3, increased the number of the resultant melanocytes in culture (Aoki et al.,2005). In contrast, the addition of antagonists for Kit or EDN3 decreased the number of the resultant melanocytes in culture.

To investigate whether the developmental pathway of the transplanted melanocytes in the iris also depended on Kit and/or EDN3 signaling, we administered ACK2 and/or BQ788, an inhibitor of EDN3, by intravitreous injection at day 2 after the transplantation of ES cell-derived melanocytes. By day 10 post transplantation, the area covered by the pigmented melanocytes was decreased by the administration of ACK2 and/or BQ788 compared with that obtained when phosphate buffered saline (PBS) was administered as a control (Fig. 4A–H), as was the total number of the pigmented melanocytes (Fig. 4I); and this reduction in number was significant. Notably, in approximately half of these eyes, we could not find any melanocytes (6/10 eyes administered ACK2, 6/10 eyes administered BQ788, and 7/12 eyes treated with both Ack2 and BQ788). In contrast, pigmented melanocytes were observed in all eyes administered PBS as a control (10/10 control eyes). These results indicate that the transplanted melanoblasts derived from ES cells proliferated and differentiated after transplantation in the host iris but not the melanocytes that already differentiated before transplantation differentiated, and further indicate that their proliferation and differentiation was occurred in a c-kit/SCF and EDNRB/EDN3-dependent manner as observed in the in vivo development and/or ES cell differentiation in vitro.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We used the iris as a recipient tissue for pigment cells, because the iris contains both melanocytes and pigmented epithelium and is easy to manipulate and suitable for observation of the transplanted cells. Our results showed that pigment cells including melanocyte and pigmented epithelial cell lineages derived from ES cells survived and differentiated when transplanted onto the iris of an allogenic host.

First, the capacity of the iris to accept exogenous pigment cells should be noted. The albino host mice used in our experiments contained unpigmented but fully differentiated melanocytes and pigmented epithelium in their iris. Despite these pre-existing melanin-lacking melanocytes and pigmented epithelium, we were able to obtain consistent and significant transfer and differentiation of melanocytes and pigmented epithelium in the adult iris, even those differentiated from ES cells in vitro. Thus, judging from the simple transplantation procedures and reproducible engraftment, the iris and the anterior chamber of the eye appear to be ideal environments for the in vivo development of not only melanocyte lineage cells as previously shown (Aoki et al.,2008) but also cells of the pigmented epithelium lineage.

The transplanted ES cell-derived melanocytes showed the dendritic morphology (Fig. 2) and spread out to cover a large area of the iris, indicating their highly migratory character as is observed with normal melanocytes from the neonatal skin. On the other hand, the pigmented epithelial cells, in both ES cell derivatives and uveal ones, showed a restricted migration after transplantation, indicating less migratory ability of this cell type versus the melanocytes. In addition, both of these pigment cells became localized to their original anatomic locations, indicating their identities to be comparable to those of their in vivo counterparts. Also, the dependency on both Kit and EDN3 signaling pathways during regenerative differentiation in the iris also indicate the identity of ES cell-derived pigmented cells as melanocytes. Thus, the ES cell-derived cells generated after transplantation to the iris were pigment cells from both morphological and cell growth requirement aspects.

When we transplanted the melanocytes derived from ES cells, we did not purify the cells used for the transplantation and we transplanted all of the cells existed in the culture system include neurons, glias, and smooth muscle cells (shown in Fig. 2A–C). Although we can observe many GFP-positive cells in the host iris at 10 day after transplantation, these GFP-positive cells are black-pigmented melanocytes with dendritic morphology (Fig. 2F–I). Tuj1-, GFAP-, and SMA-positive cells did not include the GFP-positive cells, suggesting that these cells are host-derived cells and the host iris provide the suitable environment for melanocyte-lineage cells derived from ES cell as well as in vivo derivatives. In addition, to distinguish whether the melanocytes differentiated after transplantation were melanocytes present before the transplantation or those that did differentiate after transplantation, we used ACK2 and BQ788, functional inhibitors for c-kit/SCF and EDNRB/EDN3 signals, respectively. The result showed a drastic decrease of the number of melanocytes in the recipient iris, indicating melanocyte differentiation after transplantation, from the early precursors of melanocytes present in the ES cell-derivatives.

In these experiments, the transplanted allogenic pigment cells were maintained for more than 30 days (data not shown). This finding might be due to the induction of anterior chamber-associated immune deviation (ACAID), including the suppression of delayed hypersensitivity and inactivation of humoral immunity and primed cytotoxic T cell responses (Streilein,1990). This is another advantage for the use of the iris as a recipient tissue for studying transplanted pigment cells in vivo. Reportedly, human uveal melanocytes are in a very quiescent state in vivo (Hu,2000). The absence of the growth and migration of the uveal melanocytes is observed even under various pathological conditions (such as inflammation and trauma), which usually result in proliferation and migration of host cells. The high level of TGF-β2 in the aqueous humor may play an important role for maintaining the nonproliferative, relatively quiescence status of uveal melanocytes in vivo and also in vitro (Hu et al.,1998). As shown here for mouse ES cell-derived melanocytes and previously for adult uveal or neonatal mouse skin melanocytes (Aoki et al.,2008), isolated and transplanted melanocytes or their precursors proliferated and differentiated in the adult mouse iris. This apparent discrepancy might have been caused by the procedure used to dissociate the cells from the tissue, thus, bringing these cells a highly proliferative state. In fact, in vitro cultured human uveal melanocytes were shown to have the ability to proliferate and produce melanin (Hu,2000). On the other hand, human and mouse epidermal melanocytes seem to maintain their activated state; these melanocytes are required to proliferate to supply melanosomes to the rapidly turning over skin keratinocytes in vivo, including the constantly regenerating hair keratinocytes. Because the regenerative turnover of the iris is not as high as that of skin, uveal melanocytes are not required to sharply respond to their environmental tissue and, thus, even in mice, uveal melanocytes might be relatively quiescent compared to epidermal melanocytes.

In contrast to the successful transplantation of not only melanocytes obtained in vivo but also ES cell derivatives obtained in vitro onto the iris in the eye, we have never succeeded in the transplantation of melanocytes into adult or neonatal cutaneous or subcutaneous skin of MHC-matched mice. The transplantation of cells of the melanocytic lineage was reported only in the case of embryonic skin (Oshima et al.,2001; Nishimura et al.,2002). However, in human beings, transplantation of autologous melanocytes for the treatment of vitiligo has been done successfully in several large series of patients and followed up for a long period (Olsson and Juhlin,2002; Chen et al.,2004). One of the reasons for this discrepancy may be the difference in the distribution of the melanocyte lineage cells in the adult cutaneous tissue. In mouse skin, melanocytes disappear from the interfollicular epidermis after around 1 week of birth and become restricted to the hair follicles (Hirobe,1984), and melanocyte stem cells are restricted to the lower permanent portion of mouse hair follicles, a site specialized for melanocyte stem cells (Nishimura et al.,2002). On the other hand, in human skin, melanocytes are maintained in the interfollicular epidermis even into adulthood. The disappearance of the melanocytes from the interfollicular epidermis in the mouse is possibly caused by the elimination of the SCF, one of the most important supporting factors for melanocyte development in the epidermis (Kunisada et al,1998). Due to the loss of the SCF signaling in the mouse epidermis, unlike in the case of human epidermis, the transplanted melanocytes could not survive in the mouse skin. Also, melanocytes are uniformly distributed in the stroma of the uveal tissues including the iris. Compared with the skin, the iris contains a dense network of blood vessels and neurons, as was shown in Figures 1J and 2G–I; and these cells may supply cytokines and matrix proteins to stimulate the growth and differentiation of transplanted pigment cells. Considering the fact that the iris can support the development of transplanted pigment cells even into adulthood, the environmental cues important for the self-renewal and differentiation of melanocytes may be present in the iris tissue.

Our model of successful transplantation of ES cell-derived melanocytes and pigmented epithelium into the iris may lead to a better understanding of the development of melanocytes and pigmented epithelium in vivo. Also, unexpectedly, the lower antigenicity of allogenic ES cells may provide a rationale to use allogenic cell sources for the treatment of the very common defects of retinal pigmented epithelium in addition to their use in a new therapeutic approach for ocular diseases caused by defective ocular melanocytes.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Animals

All animal experiments were approved by the Animal Research Committee, Graduate School of Medicine, Gifu University. Seven-week-old male ICR mice were obtained commercially (Chubu Kagakusizai, Co., Japan) as the recipients of the allogenic transplantation. Also used in this study were C57BL/6-background GFP mice (RIKEN, BRC) and C57BL/6-background DCT-LacZ mice (Mackenzie et al.,1997). They were housed in a standard animal room with food and water under controlled humidity and temperature (22°C ± 2°C). The room was illuminated by fluorescent lights that were on from 8:00 AM to 8:00 PM.

Cell Culture

Establishment of the EGFP-expressing D3 ES cell line and Dct-LacZ ES cell line were described earlier (Hirano et al.,2003). ES cells and the PA6 and ST2 stromal cell lines were maintained and prepared for in vitro differentiation as described (Hirano et al.,2003; Aoki et al.,2007a; Motohashi et al.,2006; Motohashi et al.,2007). For differentiation, 1,000 trypsinized ES cells were inoculated into six-well plates previously seeded with PA6 (for RPE cells) or ST2 cells (for melanocytes), and induced to differentiate in α-Minimal Essential Medium (Invitrogen, CA) supplemented with 10% fetal calf serum (FCS, EQUITECH-BIO, Inc., TX) under 5% CO2 at 37°C. Unless otherwise mentioned, incubation with 40 pM fibroblast growth factor 2 (FGF2, R&D Systems, Inc., Minneapolis, MN) and 10 nM dexamethasone (Dex, Sigma-Aldrich, St. Louis, MO) was started at day 0 and day 3, respectively; and that with 10 pM cholera toxin (CT, Sigma) was done from day 0 to 3 only for induction of the eye-like structures. The following factors were added for induction of melanocytes: 10 nM Dex, 20 pM FGF2, and 10 pM CT, as reported earlier (Aoki et al.,2005,2006). The medium was changed twice a week.

Cell Preparation

Day-19 cultured ES cell-derived melanocytes were dissociated with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA; Invitrogen) 37°C for 5 min. Day-10 cultured ES cell-derived eye-like structures including black-pigmented epithelial cells were picked up and collected separately in 0.25% trypsin/EDTA supplemented with 0.1% collagenase type 4. They were dissociated with 0.25% trypsin/EDTA 37°C for 10 min. The cells were then washed 3 times with Dulbecco's Modified Eagles Medium (DMEM, Invitrogen) supplemented with 10% FCS. The cells from each source were then centrifuged, and each was concentrated at 10,000 cells/μl in PBS.

Cells prepared from postnatal day 0 (P0) EGFP mouse epidermis were prepared according to a previously described method (Kishimoto et al.,1999; Aoki et al.,2008). Pigmented epithelium from P0 EGFP mice was also prepared according to a previously described method with minor modifications (Aoki et al.,2006). Briefly, eyes were enucleated, and the cornea and lens were removed. Then the eyes were treated with 0.25% trypsin/EDTA 4°C overnight. Then the iris and ciliary body were collected in DMEM supplemented with 10% FCS. The neural retina was removed from the RPE and underlying choroid and sclera, and then the RPE excluding the peripheral area and that around the optic nerve was peeled off from the sclera by tearing it off little by little. The RPE, thus obtained, was immersed in DMEM supplemented with 10% FCS and dissociated into single cells by gentle pipetting. After centrifugation, the cells were then resuspended at a concentration of 10,000 cells/μl in PBS.

Percentage of Melanocytes and Melanoblasts Induced From ES Cells

To estimate the fraction of pigmented malanocytes, blackly pigmented melanocytes were counted under a microscope. To estimate the fraction of melanoblasts, Dct-LacZ ES cells allowing expression of LacZ under the control of melanoblast-specific Dct promoter were used. In this case, cells were fixed and stained with X-gal on six-well plates and the number of LacZ-positive cells was counted under a microscope. To determine the total cell number, cells were trypsinized and counted with a hemocytometer. On day 19, approximately 0.13% of total cells become blackly pigmented melanocytes; calculated from 10 wells in which an average of 6,527 ± 240 pigmented cells were detected in a total of 5.0 × 106 ± 4.9 × 105 cells per well. In the separate experiment using Dct-LacZ ES cells, 0.81% were LacZ-positive melanoblasts; calculated from 5 wells in which an average of 35,628 ± 6,346 melanoblasts were detected in a total of 4.4 × 106 ± 4.0 × 105 cells per well. LacZ staining was performed as described in Histology section.

Transplantation Procedures

Surgical procedures for the transplantation were described elsewhere in detail, (Timmers et al.,2001; Aoki et al.,2007b) and were used in this study with some modifications. Briefly, mice were anesthetized with an intraperitoneal injection of ketamine (70 mg/kg; Pfizer, NY) and xylazine (14 mg/kg; Bayer, Germany). The animals were then placed in the prone position under an SN MD-II operation microscope (Nagashima Medical Instruments Co, LTD, Japan). Next, the pupils were dilated with 1 drop of 0.5% phenylephrine hydrochloride-0.5% tropicamide solution (Mydrin P, Santen, Japan), and 0.4% oxybuprocaine (Benoxil, Santen) was then applied as a topical anesthetic. The ocular fundus was visualized by the application of 1.5% hydroxycellulose (SCOPISOL15, Senju, Japan) to the eye. The cornea was then carefully punctured nasally, approximately 0.5 mm medial to the dilated papillary margin, by using a 30-gauge hypodermic needle (Becton Dickinson & Company, NJ) to allow the aqueous humor to flow out and the anterior chamber to collapse. This needle was then advanced through the cornea into the anterior chamber parallel to the anterior face of the lens, after which 1 μl of hyaluronate sodium (Healon, Pfizer or VISCORT, Alcon, TX) was injected for the purpose of preventing the outflow of the transplanted cells and making the space between the lens or iris and cornea. A 33-gauge blunt needle (Hamilton Company, Reno, NV) was next inserted through this corneal puncture and advanced into the anterior chamber, avoiding trauma to the iris and lens. Subsequently, the needle shaft was directed toward the iris.

Melanocytes were transplanted into the iris from a single injection site into one area, whereas the pigmented epithelial cells were transplanted into another iris from a single injection site into the multiple areas. Once the tip of the needle had reached the desired location for transplantation, 2 μl of cell suspension for melanocytes and 1 μl of cell suspension per point for pigmented epithelial cells were injected. We took care not to break blood vessels on the iris to prevent bleeding.

In the inhibitor experiment, at 2 days after transplantation of melanocytes, 2 μl of 40 mg/ml ACK2 in PBS (Nishikawa et al.,1991) and/or 400 μg/ml of BQ788 (Peptide Institute, Inc., Louisville, KY) or 2 μl of PBS (vehicle only) as a control were administered into the vitreous cavity by using the same protocol shown above.

Histology

For immunocytochemistry, ES cell-derived melanocytes and eye-like structures in vitro were fixed in 4% paraformaldehyde in PBS for 15 min, washed in PBS, made permeable by immersion in 0.1% Triton X-100, 0.5% BSA, and 2% skim milk in PBS for 30 min, and then washed in PBS. They were then incubated for 1 hr at room temperature with the desired primary antibody diluted in 0.5% BSA in PBS. After having been washed in PBS, the cells were stained with the appropriate secondary antibody in the same manner.

The methods used for the immunohistochemistry of the host iris were described previously (Aoki et al.,2007b). Briefly, 10 days after the transplantation, the mice were euthanized with an overdose of pentobarbital sodium. The eyes were then enucleated and fixed by immersion in 10% formalin in phosphate buffer (pH 7.2) overnight. Then the irises were picked out and processed by the same methods used for cell culture as described above. For immunohistochemistry of prepared sections, eyes were dehydrated with ethanol and embedded in paraffin. Serial sections of 3-μm thickness were cut, deparaffinized, and stained immunohistochemically or with hematoxylin and eosin (H&E).

LacZ staining was performed as reported in detail (Yoshida et al.,1996). In brief, eyes were fixed for 30 min in 4% paraformaldehyde and PBS (1:1) supplemented with 0.2% glutaraldehyde and 0.02% Tween20. After 3 washes in PBS, the eyes were stained overnight in 1.0 mM MgCl2 in 10 mM phosphate buffer (pH 7.2) containing 3.1 mM K4[Fe(CN) 6] and 3.1 mM K3[Fe(CN) 6] with 0.2 mg/ml X-Gal at 37°C. The staining reaction was stopped by washing in PBS. The specimens were post-fixed overnight in 10% formalin in phosphate buffer (pH 7.2).

Cell Counting and Statistical Analysis

At 10 days after transplantation of melanocytes, the numbers of the melanocytes in the surgically removed iris tissues was counted under a stereomicroscope. The iris tissues from the 10 eyes administered ACK2, 10 eyes with BQ788, 12 eyes treated with both Ack2 and BQ788, and 10 eyes administered PBS as control were examined. Values were expressed as the means ± SEM. The statistical significance of difference was determined by analysis of variance followed by Tukey's test. Probability values P < 0.01 were considered significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Kyoko Takahashi and Kazuko Goto for their excellent technical assistance and advice. We also thank Drs. Masayuki Niwa, Yasuhiro Yamada, Shigeru Kinoshita, and Hisato Kondo for their thoughtful advice.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
FilenameFormatSizeDescription
SuppFigS1.TIF278KSupp Fig S1. Spatiotemporal <I>Sfrp2</I> distribution in developing mouse limbs. Whole-mount <I>Sfrp2</I> expression of forelimbs and hindlimbs. (a) 10.5 d.p.c. (b) 11.5 d.p.c. (c) 12.5 d.p.c. (d) 13.5 d.p.c. (e) 14.5 d.p.c.
SuppFigS2.TIF393KSupp Fig S2. Computational merge of <I>Sfrp2</I> and Alcian blue staining. <I>Progenesis</I> software was used. (a) 12.5 d.p.c. (b) 13.5 d.p.c. (c) 14.5 d.p.c. Red colour represents alcian blue staining positive areas and green colour represents <I>Sfrp2</I> expression.
SuppFigS3.TIF471KSupp Fig S3. Section in situ analysis of (a) Sfrp1, (b) <I>Wnt4</I>, (c) <I>Sfrp2</I>, (d) <I>Gdf5</I>, (e) <I>Wnt9a</I>, and (f) <I>Sfrp4</I> in developing limbs of wild-type C57BL/6 mice. E15.0, bilateral hindlimbs are shown. Serial sections from the same limbs were analyzed.

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