Epithelial–Mesenchymal Transition in Colonies of Rhesus Monkey Embryonic Stem Ce - 《stemcells》2004-2005文献干细胞之家



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a Institut für Anatomie, Lehrstuhl für Anatomie und Entwicklungsbiologie, Universit?tsklinikum Essen, Essen, Germany;9 |& M0 e* t3 T" l9 z4 I7 |; W

b Zentrum Anatomie, Abteilung Anatomie/Embryologie, Universit?t G?ttingen, G?ttingen, Germany;
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c Stiftung caesar, Bonn, Germany& q* G% T7 `9 ~9 C. J& d2 r

Key Words. Embryonic stem cell ? Rhesus monkey ? Primate ? Differentiation ? Epithelial–mesenchymal transition ? Gastrulation ? Brachyury ? Snail2 ? E-cadherin

Correspondence: Hans-Werner Denker, Prof. Dr. Dr., Institut für Anatomie, Lehrstuhl für Anatomie und Entwicklungsbiologie, Univer-sit?tsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. Telephone: 49-201-7234380; Fax: 49-201-7235916; e-mail denker@uni-essen.de

ABSTRACT9 c' Q+ l; b3 {/ Y3 B

Embryonic stem (ES) cell lines have been established from cultures of primate embryos, including rhesus , marmoset , and cynomolgus monkeys  as well as humans . Since then, a large number of studies have found that these cells are capable of differentiating into almost all of the specialized cells of the body and, thus, may have the potential to generate replacement cells for a wide range of tissues and organs.
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The ability to give rise to differentiated cell types that are derived from all three primary germ layers of the embryo〞that is, endoderm, mesoderm and ectoderm〞makes ES cells unique. However, our understanding of the developmental potential of ES cells is still fragmentary. Indeed, mouse ES cells have a pattern formation potential that has so far only rarely been studied in detail . Although mouse embryoid bodies only partially mimic the early embryonic body plan formation process, particularly striking patterning processes were reported to occur in marmoset monkey ES cell colonies. When grown at high densities, these ES cells have been reported to form embryoid bodies, which, interestingly, have been described to show a close resemblance to early postimplantation embryos, including the formation of a yolk sac, amnion, and embryonic disc with an early primitive streak . Although the identity of the component cells of these structures has not been demonstrated conclusively, the observation has led to the assumption that non-human primate stem cells might be good model system for early primate development .

ES cells derived from the common marmoset  are characterized by a very long replication time and, in addition, a pronounced tendency to differentiate and stop dividing. This makes it difficult to keep them in cell culture for many passages (Thomson, personal communication and unpublished observations) and makes it impossible to obtain sufficient amounts of cells for extensive analyses. Marmoset ES cells are for this reason not commercially available anymore. Therefore, we chose to use the only available rhesus monkey embryonic stem (rhES) cell line, R366.4 , to further analyze the differentiation and pattern formation potential of primate ES cells in vitro. Here, we show that under the conditions chosen, these rhES cells can differentiate in vitro into complex structures in which a process can regularly be observed that, according to morphological and molecular characteristics, seems to mimic the epithelial–mesenchymal transition (EMT) as taking place during gastrulation.: n; h8 I. J  o) `  c3 c, K
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MATERIALS AND METHODS
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General Morphology7 Y7 {5 N( e3 X# F6 Q5 _
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Confluent monolayers of MEFs were cultured on gelatin-coated tissue culture dishes and used as feeder cells to grow the rhES cell line R366.4 for up to 10 days. During the day after seeding, ES cells attached to the free surface of the mouse feeder and formed small colonies as previously described . Growth and development of ES cells were monitored daily.( O) o4 \5 C+ L( ]8 l& u* {

Over the first days, stem cell colonies increased in size and cell number and became more compact. After 3–4 days of continuous growth, colonies differentiated into more complex structures, i.e., they became roundish and flattened but multilayered. Using phase-contrast microscopy, after 3–4 days of culture, pit-like depressions became visible in the centers of almost all colonies.

Typically, the disc-like colony was multilayered, i.e., it was composed of an upper and a lower layer, and between both ES cell layers the layer of MEF cells was positioned (Fig. 1). At the periphery of the colony, the upper and the lower ES cell layers were completely separated by the MEF layer〞that is, one sheet of cells was growing on top of the feeders, thereby covering the latter, and the other was growing below the feeder cell layer, thereby fixing the colony to the culture dish (Figs. 1A–1F, 1K). However, at the described pit in the center of the colony, the upper and the lower ES cell layers were in continuity〞that is, not separated by the feeder cells (Figs. 1D, 1E, 1J). The diameters of the pits were varying between the colonies. Detailed histological examination of the centers clearly revealed that the mouse feeder cells were completely absent from these areas of the colonies (Figs. 1H–1J). The feeder layer between the ES cell layers thickened toward the periphery to reach its normal dimension as outside of the colony (Figs. 1J, 1K). Within the developing pit, the cells from the upper layer appeared to move while still in contact with their neighbors, through the feeder cell layer into the space between the feeder cells and the culture dish, thereby forming the lower layer (Figs. 1H–1K). The pit diameter increased during continuing growth of the colony. In widened flat-bottomed pits, epithelial cells obviously derived from the upper layer formed the bottom of the pit (Figs. 1D, 1E, 1J). In such enlarged pits, feeder cells had completely disappeared. Mixing of cells from the upper layer or from the lower layer of ES cells with those from the mouse feeder layer was never observed.

Figure 1. A typical differentiated colony of rhesus monkey embryonic stem cells after 5 days of culture in a series of cross-sections starting from the (A) periphery of the colony and continuing toward the (E) center. When viewed from the side, the differentiated colony is comprised of an upper and a lower layer and, between the two, the (A) preexisting layer of mouse feeder cells. (E): Note the lack of the feeder layer in the central region of the colony where the pit-like structure emerges. (F–K): Enlargements of the boxed areas in A–E. They show details of the arrangement of stem cells and feeder cells. Arrowheads indicate mouse feeder layer; asterisks, mouse embryonic fibroblasts embedded within the matrix of the feeder layer; arrows, stem cells from the upper and the lower layer, respectively, located at the periphery of the colony. Abbreviations: cs, coverslip; me, medium.

This regular morphology of the colony with a central pit was observable for only a relatively short period of approximately 2 days until colonies grew together and proliferated or migrated in a more uncoordinated fashion.
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Ultrastructural Features+ m/ j4 T) A/ C8 g* L) `! \) U9 ]

Cells of the upper layer of the colonies exhibited epithelial-like characteristics with specialized cell-cell junctions anchored by prominent bundles of filaments in their apical cell pole (Figs. 2A, 2C). These cells were clearly polarized, with their tight junctions being located in the subapical region while the remainder of the lateral plasma membrane showed only few punctual adherens junctions, and here the intercellular space was wide. The apical surface of cells showed microvilli-like structures as well as plump cytoplasmic protrusions. The cells had a low nucleus-to-cytoplasm ratio and prominent nucleoli. Most of these cells showed a polarized organization of the cytoplasm, organelles being concentrated in the apical part of the cell and the nucleus close to the basal pole. Via their basal pole, almost all cells were in direct contact with a supporting matrix provided by the feeder cell layer (Fig. 2A). Electron microscopically, this matrix showed collagen fibrils, but the composition of this matrix was not analyzed in further detail. No electron-dense structure resembling a basement membrane was found.2 B' @2 v2 i% Y2 L

Figure 2. Electron micrographs of a differentiated rhesus monkey embryonic stem cell colony. Cells within the (A, C) upper layer are epithelioid, whereas (D) lower layer cells have a mesenchyme-like phenotype. The epithelioid cells are apico-basally polarized, showing specialized cell–cell junctions in their apical pole (C, inset in A). The depressed region of the central part of the colony (pit) is several cells thick, and (B) only the uppermost cells are connected by tight junctions as is typical for an epithelium. (E): As observed in the lateral region of the central pit, cells from the upper layer seem to undergo a modulation of their phenotype, to ingress, and to migrate downward to form the lower layer. Asterisk indicates mouse feeder layer; arrowheads, prominent bundles of filaments in epitheloid cells; arrows, specialized cell–cell junctions between epitheloid cells; O, intercellular space within the colony; #, epithelioid cell; , migrating mesenchyme-like cell ingressing from the upper layer. Abbreviation: me, medium.
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While the epithelial cells of the upper layer of the colony were usually attached to the feeder layer, cells within the pit lacked this support layer. Cells in the pit were usually multilayered, but typically only the upper cells facing the medium were tightly connected with each other via tight junctions, thus clearly exhibiting an epithelial phenotype. The cells of the lower layer were attached to each other via primitive punctate membrane contacts, thereby forming a loose network. These cells developed long extrusions that reached into the space surrounding the cells (Fig. 2B). The lowest cells constituting the bottom of the pit had a broad contact area with the matrix of the culture dish.

Outside the pit, the ES cells of the lower layer of the colony had a mesenchymal phenotype (Figs. 2D, 2E). They exhibited a high nucleus-to-cytoplasm ratio, were irregular in shape (polygonal), and lacked regular cell-cell contacts and apico-basal polarity. In the vicinity of the pit, cells looked scattered, suggesting that cells had acquired here a migratory phenotype (Fig. 2E). Probably these cells had moved down from the upper layer and continued now to migrate into the space between the feeder cell layer and the culture dish, thereby forming the pit-like structure of the colony.
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Distribution of ZO-1, E-cad, and Cx43
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As mentioned above, epithelial-like cells were formed in the upper layer of differentiated stem cell colonies. Here, we further analyzed the nature of specialized junctions between these cells. Of particular interest are occluding junctions (tight junctions) as they are typical of epithelial cells. Occluding junctions seal cells together in an epithelial sheet. Moreover, we characterized E-cad–mediated cell–cell adhesion, which is strictly regulated during mesoderm formation , and also the communicating junctions, which mediate signals from one cell to its interacting partner. As marker molecules for these different types of junctions, we have chosen the tight junction protein ZO-1 (Figs. 3A–3F), the epithelium-specific cell–cell adhesion protein E-cad (Figs. 3G–3L) , and the gap junction proteins Cx43 and Cx32, the latter being markers for epiblast and hypoblast, respectively  (Figs. 4A–4F).

Figure 3. Typical localization of (A–F) the tight junction protein ZO-1 and the (G–M) cell–cell adhesion protein E-cad together with (D–F, J–M) F-actin in differentiated colonies of rhesus monkey embryonic stem cells as shown by confocal laser-scanning microscopy. Optical sections are from (A, D, G, J) the upper layer of the colony, grazing the (B, E; H, K) pit in the center of the colony and the (C, F; I, L) lower layer. Note the web-like staining pattern of both ZO-1 and Ecad in the upper layer (white arrows) and the top cells in the pit (black arrows), respectively. In contrast, cells of the lower layer showed only weak and rather irregular staining of ZO-1 and E-cad (asterisks). M, An E-cad stained colony in an xz-section. Green staining representing E-cad is much stronger in the upper layer compared with the lower layer of the colony. Abbreviations: CS, coverslip; E-cad, E-cadherin.

Figure 4. (A–F): Typical localization of the gap junction protein connexin Cx43 together with (D–F) F-actin in differentiated colonies of rhesus monkey embryonic stem cells as shown by confocal laser-scanning microscopy. (A, D): Optical sections are from the upper layer of the colony, grazing the (B, E) pit in the center of the colony and the (C, F) lower layer. Note the web-like staining pattern of Cx43 in the upper layer (white arrows) and the top cells in the pit (black arrows). In contrast, cells of the lower layer showed only weak and rather irregular staining of Cx43 (asterisks).9 T/ n7 [4 l. d
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ZO-1 immunohistochemistry revealed that the upper layer (Figs. 3A, 3D) but not the lower layer (Figs. 3C, 3F) was comprised of ZO-1–positive cells. The tight junction protein ZO-1 was found at the subapical part of the lateral border of adjacent cells in the top layer cells, as were tight junctions in transmission electron microscopy (see Fig. 2). In contrast, ZO-1 was only very weakly detectable in cells of the lower layer (Figs. 3C, 3F). E-cad was found to be strongly expressed in adhesion belts of the epithelial cells in the top layer (Figs. 3G, 3J, 3M) and in those cells lining the pit in the center of the colonies (Fig. 3H, 3K). In contrast, E-cad largely disappeared in the cells of the lower layer (Figs. 3I, 3L, 3M). This change of marker expression correlates well with the morphological changes observed in these cells (see Figs. 1, 2). Thus, the upper layer cells that are covering the surface of the colony can be characterized as an epithelium, including those cells that were lining the pit in the center of the colonies (Figs. 3B, 3E, 3H, 3K), creating a seal that isolates the interior of the colony from the external medium.$ a% C9 ?5 Z  q- k

Upper layer cells also were coupled via gap junctions, as suggested by their expression of connexin Cx43 (Figs. 4A–4F). Epithelial cells of the outermost layer in the periphery (Figs. 4A, 4D) as well as in the center of the pits (Figs. 4B, 4E) showed a clearly positive immunohistochemical signal for Cx43. Confocal microscopy revealed a web-like pattern, suggesting that gap junction protein Cx43 was localized within lateral membrane domains of adjacent cells. With respect to the mesenchyme-like cells of the lower layer, Cx43 fluorescence was significantly weaker in many places (Figs. 4C, 4F), and instead of web-like patterns, weak spotty signals were found irregularly distributed within the cells. In contrast to Cx43, Cx32 was observed neither in cells of the upper layer nor in those of the lower layer, whereas monkey liver exhibited a strong and specific signal (positive control; data not shown).

Expression of ES Cell–Specific Markers% [! f/ Z" Z. g/ @  [& v( ?
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Established ES cell markers include alkaline phosphatase, the POU domain gene product Oct-4, and telomerase reverse transcriptase , all known to be involved in or associated with pluripotency of embryonic stem cells; i.e., their expression was reported to decrease upon stem cell differentiation. After forming morphologically differentiated colonies, rhES cells still expressed all markers tested, as revealed by histochemistry (alkaline phosphatase, Fig. 5A), in situ hybridization (Oct-4, Fig. 5B), and reverse transcription–PCR (Oct-4 and Tert, Figs. 5C, 5D). Interestingly, the mRNA levels of Tert strongly decreased between days 2 and 4 of the culture and remained low thereafter. In contrast, the expression of Oct-4 continuously increased during the differentiation of the colonies. At day 10, the mRNA abundance of Oct-4 was approximately 2.3-fold higher than in day-2 colonies. This shows that the expression of Oct-4 and Tert, both reported to be markers for pluripotent stem cells, diverges in differentiating rhES cell colonies. Interestingly, in situ hybridization revealed that almost all cells within a colony contained Oct-4 mRNA, although at the stage of colony development shown in Figure 5B, a large subpopulation of cells in the colony had already differentiated to epithelial cells (compare with Figs. 3, 4).# ~9 {% F: F" S0 \5 t

Figure 5. Full-face view of differentiated colonies of rhesus monkey embryonic stem (rhES) cells after (A) alkaline phosphatase staining and (B) whole-mount in situ hybridization with Oct-4, respectively. Alkaline phosphatase and Oct-4 are detectable in almost all cells of the colony. The upper layer of the colony is marked (arrows in A). (C, D): Quantification of undifferentiated stem cell marker mRNA expression in rhES cells after 2, 4, 6, 8, and 10 days of culture. (C): The mRNA for the transcription factor Oct-4 was clearly upregu-lated, whereas the telomerase reverse transcriptase (Tert) mRNA was downregulated during the experimental period. (D): Quantification of the signals shown in (C). The value determined for day 2 was set as 1. All other values given are relative to this time point. Abbreviation: MEF, mouse embryonic fibroblast.) C' w8 H: C- ~4 H  q3 O4 S

Expression of Developmental Marker Genes

Since morphological observations made in marmoset monkey ES cell colonies  had suggested that primitive streak equivalents can form there, we wished to obtain information on EMT- and gastrulation-related gene expression in differentiating rhES cell colonies. This was done by in situ hybridization (Fig. 6) and quantitative reverse transcription–PCR (Fig. 7) on cDNA isolated from colonies of different developmental stages. The transcription factor genes Brachyury, goosecoid, Snail2, and MesP2 (primate MesP2 is more closely related to mouse MesP1 than primate MesP1 and was therefore investigated in this study) were investigated as markers for gastrulation. Brachyury is a reliable marker for mesoderm progenitor cells in normal embryos. Its expression is induced in epiblast cells of the rabbit several hours before the first visible signs of primitive streak formation and EMT . Brachyury was strongly expressed in our experimental system in day-2 colonies as revealed by reverse transcription–PCR (Fig. 7). During further differentiation of the colonies, the expression level of Brachyury continuously and dramatically decreased. At day 10, there was almost no Brachyury mRNA detectable. In the rhES cell colonies, goosecoid mRNA abundance increased between days 2 and 4. Thereafter, goosecoid signal intensity clearly decreased and dropped to low levels at day 10 (Fig. 7). In contrast to Brachyury and goosecoid, Snail2 (or Slug), a gene specifically expressed in the embryo in early mesodermal cells emigrating from the primitive streak and undergoing an EMT , was more than 10-fold upregulated between days 2 and 6. MesP1 expression in the mouse embryo was first observed at the onset of gastrulation in the nascent mesodermal cells that first ingressed at the end of the primitive streak . The abundance of MesP2 mRNA increased approximately twofold in the rhES cell colonies between days 2 and 6. E-cad mRNA signal increased between days 2 and 4, remained high until day 8, and slightly declined thereafter. This may reflect the formation of epithelial sheets followed by a relative decrease of epithelial cells during formation of lower-layer mesenchymal cells at the later stages.! ~8 V% h1 Y! I; {* }; ^, t7 @
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Figure 6. (A): Brachyury and (B) goosecoid expression in differentiated colonies of rhesus monkey embryonic stem cells as revealed by in situ hybridization (full-face view). (C): Cellular distribution of Brachyury expression as seen in cross sections. Note that Brachyury transcripts are not restricted to a specific layer or cell population of the colony but are found in all cells irrespective of their position. Mouse embryonic feeder cells show no staining.
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Figure 7. Quantification of marker mRNA expression in rhesus monkey embryonic stem cells after 2, 4, 6, 8, and 10 days of culture. (A): The mRNAs for the transcription factors Brachyury and goosecoid are clearly downregulated during the experimental period. In contrast, the transcription factors Snail2 and MesP2 are upregulated during the first days of culture, whereas E-cad signal intensity increases until day 4 and slightly decreases at day 10. (B): Quantification of the signals shown in (A). The value determined for day 2 was set as 1. All other values given are relative to this time point. All values were normalized to ?-actin. Abbreviations: E-cad, E-cadherin; MEF, mouse embryonic fibroblast.# _+ p( Q) R7 f4 |% C' J( I* O

In situ hybridization on whole differentiated colonies from day 4 with probes specific to Brachyury and goosecoid revealed that both marker genes were detectable in the cytoplasm of both cell layers of the colonies (Fig. 6C). However, a pattern or confinement of the signal to single cells or clusters of cells within the colonies could not be observed. Specificity of the hybridization signal was shown by the fact that neither the nuclei of the ES cells nor the mouse feeder cells showed any significant staining with the antisense probe (Fig. 6C). Moreover, the sense controls showed almost no staining.

DISCUSSION

R.B. and M.T. contributed equally to this study. We thank Prof. Dr. A. Wobus for generous support and advice. The skillful technical assistance of I. Kromberg, B. Maranca-Hüwel, D. Schünke, B. Gobs-Hevelke, and R. Brand is acknowledged with great gratitude. This study was supported in part by a grant from the Interne Forschungsf?rderung Essen (IFORES) to R.B. and a grant from the Kompetenznetzwerk Stammzellforschung NRW to M.T. and H.W.D.6 P* ]8 Q$ o. p

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